Microfluidic method for nucleic acid purification and processing

ABSTRACT

Integrated microfluidic devices comprising at least an enrichment channel ( 10 ) and a main electrophoretic flowpath ( 12 ) are provided. In the subject integrated devices, the enrichment channel and the main electrophoretic flowpath are positioned so that waste fluid flows away from said main electrophoretic flowpath through a discharge outlet ( 6 ). The subject devices find use in a variety of electrophoretic applications, including clinical assays, high throughput screening for genomics and pharmaceutical applications, point-or-care in vitro diagnostics, molecular genetic analysis and nucleic acid diagnostics, cell separations, and bioresearch generally.

This is a Continuation application of prior application Ser. No.09/018,918 filed on Feb. 5, 1998, now U.S. Pat. No. 6,074,827 disclosureof which is incorporated herein by reference.

This application is a Continuation-in-part of U.S. Ser. No. 08/982,855,filed Dec. 2, 1997, now U.S. Pat. No. 6,032,694, which is aContinuation-in-Part of U.S. Ser. No. 08/690,307, filed Jul. 30, 1996now U.S. Pat. No. 5,770,029.

BACKGROUND

This invention relates to microfluidics, and particularly tomicrochannel devices in which fluids are manipulated at least in part byapplication of electrical fields.

Electrophoresis has become an indispensable tool of the biotechnologyand other industries, as it is used extensively in a variety ofapplications, including the separation, identification and preparationof pure samples of nucleic acids, proteins, carbohydrates, theidentification of a particular analyte in a complex mixture, and thelike. Of increasing interest in the broader field of electrophoresis iscapillary electrophoresis (CE), where particular entities or species aremoved through a medium in an electrophoretic chamber of capillarydimensions under the influence of an applied electric field. Benefits ofCE include rapid run times, high separation efficiency, small samplevolumes, etc. Although CE was originally carried out in capillary tubes,of increasing interest is the practice of using microchannels ortrenches of capillary dimension on a planar substrate, known asmicrochannel electrophoresis (MCE). CE and MCE are increasingly findinguse in a number of different applications in both basic research andindustrial processes, including analytical, biomedical, pharmaceutical,environmental, molecular, biological, food and clinical applications.

Despite the many advantages of CE and MCE, the potential benefits ofthese techniques have not yet been fully realized for a variety ofreasons. Because of the nature of the electrophoretic chambers employedin CE and MCE, good results are not generally obtainable with sampleshaving analyte concentrations of less than about 10⁻⁶ M. This loweranalyte concentration detection limit has significantly limited thepotential applications for CE and MCE. For example, CE and MCE have notfound widespread use in clinical applications, where often an analyte ofinterest is present in femtomolar to nanomolar concentration in acomplex sample, such as blood or urine.

In order to improve the detection limits of CE, different techniqueshave been developed, including improved sample injection procedures,such as analyte stacking (Beckers & Ackermans, “The Effect of SampleStacking for High Performance Capillary Electrophoresis,” J. Chromatogr.(1993) 629: 371-378), field amplification (Chien & Burgi, “FieldAmplified Sample Injection in High-Performance CapillaryElectrophoresis,” J. Chromatogr. (1991) 559: 141-152), and transientisotachophoresis (Stegehuis et al., “Isotachophoresis as an On-LineConcentration Pretreatment Technique in Capillary Electrophoresis,” J.Chromatogr. (1991) 538: 393-402), as well as improved sample detectionprocedures and “off-line” sample preparation procedures.

Another technique that has been developed to improve the detection limitachievable with CE has been to employ an analyte preconcentration devicethat is positioned directly upstream from the capillary, i.e., in an“on-line” or “single flow path” relationship. As used herein, the term“on-line” and “single flow path” are used to refer to the relationshipwhere all of the fluid introduced into the analyte preconcentrationcomponent, i.e., the enriched fraction and the remaining waste fractionof the original sample volume, necessarily flows through the mainelectrophoretic portion of the device, i.e., the capillary tubecomprising the separation medium. A review of the various configurationsthat have been employed is provided in Tomlinson et al., “Enhancement ofConcentration Limits of Detection in CE and CE-MS: A Review of On-LineSample Extraction, Cleanup, Analyte Preconcentration, and MicroreactorTechnology,” J. Cap. Elec. (1995) 2: 247-266, and the figures providedtherein.

Although this latter approach can provide improved results with regardto analyte detection limits, particularly with respect to theconcentration limit of detection, it can have a deleterious impact onother aspects of CE, and thereby reduce the overall achievableperformance. For example, analyte peak widths can be broader in on-lineor single flow path devices comprising analyte preconcentrators.

Accordingly, there is continued interest in the development of improvedCE devices capable of providing good results with samples having lowconcentrations of analyte, particularly analyte concentrations in thefemtomolar to nanomolar range.

MCE devices are disclosed in U.S. Pat. Nos. 5,126,022; 5,296,114;5,180,480; 5,132,012; and 4,908,112. Other references describing MCEdevices include Harrison et al., “Micromachining a MiniaturizedCapillary Electrophoresis-Based Chemical Analysis System on a Chip,”Science (1992) 261: 895; Jacobsen et al., “Precolumn Reactions withElectrophoretic Analysis Integrated on a Microchip,” Anal. Chem. (1994)66: 2949; Effenhauser et al., “High-Speed Separation of AntisenseOligonucleotides on a Micromachined Capillary Electrophoresis Device,”Anal. Chem. (1994) 66:2949; and Woolley & Mathies, “Ultra-High-Speed DNAFragment Separations Using Capillary Array Electrophoresis Chips,”P.N.A.S. USA (1994) 91:11348.

Patents disclosing devices and methods for the preconcentration ofanalyte in a sample “on-line” prior to CE include U.S. Pat. Nos.5,202,010; 5,246,577 and 5,340,452. A review of various methods ofanalyte preconcentration employed in CE is provided in Tomlinson et al.,“Enhancement of Concentration Limits of Detection in CE and CE-MS: AReview of On-Line Sample Extraction, Cleanup, Analyte Preconcentration,and Microreactor Technology,” J. Cap. Elec. (1995) 2: 247-266.

SUMMARY OF THE INVENTION

Integrated electrophoretic microdevices comprising at least anenrichment channel and a main electrophoretic flowpath, as well asmethods for their use in electrophoretic applications, are provided. Theenrichment channel serves to enrich a particular fraction of a liquidsample for subsequent movement through the main electrophoreticflowpath. In the subject devices, the enrichment channel andelectrophoretic flowpath are positioned such that waste fluid from theenrichment channel does not flow through the main electrophoreticflowpath, but instead flows through a discharge outlet. The subjectdevices find use in a variety of electrophoretic applications, whereentities are moved through a medium in response to an applied electricfield. The subject devices can be particularly useful in high throughputscreening, for genomics and pharmaceutical applications such as genediscovery, drug discovery and development, and clinical development; forpoint-of-care in vitro diagnostics; for molecular genetic analysis andnucleic acid diagnostics; for cell separations including cell isolationand capture; and for bioresearch generally.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 provides a diagrammatic view of an enrichment channel for use ina device according to the subject invention.

FIG. 2 provides a diagrammatic view of an alternative embodiment of anenrichment channel also suitable for use in the subject device.

FIG. 3A provides a top diagrammatic view of a device according to thesubject invention.

FIG. 3B provides a side view of the device of FIG. 3A.

FIG. 4 provides a diagrammatic top view of another embodiment of thesubject invention.

FIG. 5 provides a diagrammatic view of an embodiment of the subjectinvention in which the enrichment channel comprises a single fluid inletand outlet.

FIG. 6 provides a diagrammatic view of a device according to the subjectinvention in which the enrichment channel comprises an electrophoreticgel medium instead of the chromatographic material, as shown in FIGS. 1and 2.

FIG. 7 provides a diagrammatic top view of disk shaped device accordingto the subject invention.

FIG. 8 is a flow diagram of a device as in FIGS. 1 or 2.

FIG. 9 is a flow diagram of a device as in FIGS. 3A, 3B.

FIG. 10 is a flow diagram of a device as in FIG. 4.

FIG. 11 is a flow diagram of a device as in FIG. 5.

FIG. 12 is a flow diagram of a device as in FIG. 6.

FIG. 13 is a flow diagram of a device as in FIG. 7.

FIG. 14 is a flow diagram of part of an embodiment of a device accordingto the invention, showing multiple inlets to the separation channel.

FIG. 15 is a flow diagram of an embodiment of a device according to theinvention, showing an alternative configuration for the intersectionbetween the main and secondary electrophoretic flowpaths.

FIG. 16 is a flow diagram of an embodiment of a device according to theinvention, showing a plurality of analytical zones arranged in seriesdownstream from the enrichment channel.

FIG. 17 is a flow diagram of an embodiment of a device according to theinvention, showing a plurality of analytical zones arranged in paralleldownstream from the enrichment channel.

FIG. 18 is a flow diagram of an embodiment of a device according to theinvention, showing a plurality of main electrophoretic flowpathsdownstream from the enrichment channel.

FIG. 19 is a flow diagram of an embodiment of a device according to theinvention, showing a plurality of enrichment channels arranged inparallel.

FIGS. 20 and 21 are flow diagrams of embodiments of a device accordingto the invention, similar to those shown in FIGS. 15 and 16,respectively, and additionally having a reagent flowpath for carrying areagent from a reservoir directly to the main electrophoretic flowpath.

FIG. 22 is a flow diagram of an embodiment of a device according to theinvention, similar to that shown in FIG. 17, respectively, andadditionally having a plurality of reagent flowpaths for carrying areagent from a reservoir directly to downstream branches of the mainelectrophoretic flowpath.

FIG. 23 is a flow diagram of an embodiment of a device according to theinvention, in which the enrichment medium includes coated magneticbeads.

FIGS. 24 and 25 are flow diagrams showing embodiments of a deviceaccording to the invention, as may be used in the DNA capture methoddescribed in Example 7.

FIG. 26 is a reaction scheme showing synthesis of the5-dethiobiotin-primer construct as described in Example 7.

FIG. 27 is a flow diagram of an embodiment of a device according to theinvention, as may be used to separate a mixture of biological entitiesinto four different subsets, by way of affinity-binding capture andrelease in affinity zones arranged in parallel.

DETAILED DESCRIPTION

Integrated electrophoretic microdevices comprising at least anenrichment channel and a main electrophoretic flowpath are provided. Theenrichment channel serves to enrich a particular analyte comprisingfraction of a liquid sample. The enrichment channel and mainelectrophoretic flowpath are positioned in the device so that wastefluid from the enrichment channel does not flow through the mainelectrophoretic channel, but instead flows away from the mainelectrophoretic flowpath through a discharge outlet. The subject devicesmay be used in a variety of electrophoretic applications, includingclinical assay applications. In further describing the invention, thedevices will first be described in general terms followed by adiscussion of representative specific embodiments of the subject deviceswith reference to the figures.

The subject device is an integrated electrophoretic microdevice. Byintegrated is meant that all of the components of the device, e.g., theenrichment channel, the main electrophoretic flowpath, etc., are presentin a single, compact, readily handled unit, such as a chip, disk or thelike. As the devices are electrophoretic, they are useful in a widevariety of the applications in which entities, such as molecules,particles, cells and the like are moved through a medium under theinfluence of an applied electric field. Depending on the nature of theentities, e.g., whether or not they carry an electrical charge, as wellas the surface chemistry of the electrophoretic chamber in which theelectrophoresis is carried out, the entities may be moved through themedium under the direct influence of the applied electric field or as aresult of bulk fluid flow through the pathway resulting from theapplication of the electric field, e.g., electroosmotic flow (EOF). Themicrodevices will comprise a microchannel as the main electrophoreticflowpath. By microchannel is meant that the electrophoretic chamber ofthe main electrophoretic flowpath in which the medium is present is aconduit, e.g., trench or channel, having a cross sectional area whichprovides for capillary flow through the chamber, where the chamber ispresent on a planar substrate, as will be described below in greaterdetail.

According to the invention the device includes an enrichment channelthat includes a sample inlet and at least one fluid outlet, and containsan enrichment medium for enriching a particular fraction of a sample;optionally, the device further includes a second fluid outlet. Thepurpose of the enrichment channel is to process the initial sample toenrich for a particular fraction thereof, where the particular fractionbeing enriched includes the analyte or analytes of interest. Theenrichment channel can thus serve to selectively separate the fractioncontaining the target analyte from the remaining components of theinitial sample volume. The target-containing fraction may be retainedwithin the enrichment channel, and the remainder flushed out from thechannel for disposal or further treatment downstream; or, alternatively,selected components may be retained within the enrichment channel, andthe target-containing fraction may be permitted to pass downstream forfurther processing.

Depending on the particular application in which the device is employed,the enrichment channel can provide for a number of different functions.The enrichment channel can serve to place the analyte of interest into asmaller volume than the initial sample volume, i.e., it can serve as ananalyte concentrator. Furthermore, it can serve to prevent potentiallyinterfering sample components from entering and flowing through the mainelectrophoretic flowpath, i.e., it can serve as a sample “clean-up”means. In addition, the enrichment channel may serve as a microreactorfor preparative processes on target analyte present in a fluid sample,such as chemical, immunological, and enzymatic processes, e.g.,labeling, protein digestion, DNA digestion or fragmentation, DNAsynthesis, and the like.

The enrichment channel may be present in the device in a variety ofconfigurations, depending on the particular enrichment medium housedtherein. The internal volume of the channel will usually range fromabout 1 pl to 1 ml, usually from about 1 pl to 100 nl, where the lengthof the channel will generally range from about 1 mm to 5 mm, usually 10mm to 1 mm, and the cross-sectional dimensions (e.g., width, height)will range from about 1 mm to 200 mm, usually from about 10 mm to 100mm. The cross-sectional shape of the channel may be circular, ellipsoid,rectangular, trapezoidal, square, or other convenient configuration.

A variety of different enrichment media may be present in the enrichmentchannel. Representative enrichment medium or means include those meansdescribed in the analyte preconcentration devices disclosed in U.S. Pat.Nos. 5,202,010; 5,246,577 and 5,340,452, as well as Tomlinson et al.,supra, the disclosures of which are herein incorporated by reference.Specific enrichment means known in the art which may be adaptable foruse in the subject integrated microchannel electrophoretic devicesinclude: those employed in protein preconcentration devices described inKasicka & Prusik, “Isotachophoretic Electrodesorption of Proteins froman Affinity Adsorbent on a Microscale,” J. Chromatogr. (1983)273:117128; capillary bundles comprising an affinity adsorbent asdescribed in U.S. Pat. No. 5,202,101 and WO 93/05390; octadodecylsilanecoated solid phases as described in Cai & El Rassi, “On-LinePreconcentration of Triazine Herbicides with Tandem OctadecylCapillaries-Capillary Zone Electrophoresis,” J. Liq. Chromatogr. (1992)15:1179-1192; solid phases coated with a metal chelating layer asdescribed in Cai & El Rassi, “Selective On-Line Preconcentration ofProteins by Tandem Metal Chelate Capillaries-Capillary ZoneElectrophoresis,” J. Liq. Chromatogr. (1993) 16:2007-2024;reversed-phase HPLC solid packing materials as described in U.S. Pat.No. 5,246,577), Protein G coated solid phases as described in Cole &Kennedy, “Selective Preconcentration for Capillary Zone ElectrophoresisUsing Protein G Immunoaffinity Capillary Chromatography,”Electrophoresis (1995) 16:549-556; meltable agarose gels as described inU.S. Pat. No. 5,423,966; affinity adsorbent materials as described inGuzman, “Biomedical Applications of On-Line Preconcentration—CapillaryElectrophoresis Using an Analyte Concentrator: Investigation of DesignOptions,” J. Liq. Chromatogr. (1995) 18:3751-3568); and solid phasereactor materials as described in U.S. Pat. No. 5,318,680. Thedisclosures of each of the above-referenced patents and otherpublications are hereby incorporated by reference herein.

One class of enrichment media or materials that may find use asenrichment media are chromatographic media or materials, particularlysorptive phase materials. Such materials include: reverse phasematerials, e.g., C8 or C18 compound coated particles; ion-exchangematerials; affinity chromatographic materials in which a binding memberis covalently bound to an insoluble matrix, where the binding member maygroup specific, e.g., a lectin, enzyme cofactor, Protein A and the like,or substance specific, e.g., antibody or binding fragment thereof,antigen for a particular antibody of interest, oligonucleotide and thelike, where the insoluble matrix to which the binding member is boundmay be particles, such as porous glass, polymeric beads, magnetic beads,networks of glass strands or filaments, a plurality of narrow rods orcapillaries, the wall of the channel and the like. Depending on thenature of the chromatographic material employed as the enrichment means,it may be necessary to employ a retention means to keep thechromatographic material in the enrichment channel. Conveniently, glassfrits or plugs of agarose gel may be employed to cover the fluid outletsor inlets of the chamber, where the frits or plugs allow for fluid flowbut not for particle or other insoluble matrix flow out of theenrichment channel. In embodiments where the enrichment means is achromatographic material, typically sample will be introduced into, andallowed to flow through, the enrichment channel. As the sample flowsthrough the enrichment channel, the analyte comprising fraction will beretained in the enrichment channel by the chromatographic material andthe remaining waste portion of the sample will flow out of the channelthrough the waste outlet.

In embodiments where the enrichment means is a bed of polymeric beads orparamagnetic beads or particles, the beads may be coated with antibodiesor other target-specific affinity binding moiety, including: affinitypurified monoclonal antibodies to any of a variety of mammalian cellmarkers, particularly human cell markers, including markers for T cells,T cell subsets, B cells, monocytes, stem cells, myeloid cells,leukocytes, and HLA Class II positive cells; secondary antibodies to anyof a variety of rodent cell markers, particularly mouse, rat or rabbitimmunoglobulins, for isolation of B cells, T cells, and T cell subsets;uncoated or tosylactivated form for custom coating with any givenbiomolecule; and streptavidin-coated for use with biotinylatedantibodies. Paramagnetic beads or particles may be retained in theenrichment channel by application of a magnetic field.

Alternatively, or in addition to solid phase materials such as coatedparticles or other insoluble matrices as the enrichment means, one mayemploy a coated and/or impregnated membrane which provides for selectiveretention of the analyte comprising fraction of the sample whileallowing the remainder of the sample to flow through the membrane andout of the enrichment means through the waste outlet. A variety ofhydrophilic, hydrophobic and ion-exchange membranes have been developedfor use in solid phase extraction which may find use in the subjectinvention. See, for example, Tomlinson et al., “Novel Modifications andClinical Applications of Preconcentration-Capillary Electrophoresis-MassSpectrometry,” J. Cap. Elect. (1995) 2: 97-104; and Tomlinson et al.,“Improved On-line Membrane Preconcentration-Capillary Electrophoresis(mPC-CE),” J. High Res. Chromatogr. (1995) 18:381-3.

Alternatively or additionally, the enrichment channel or the enrichmentmedium can include a porous membrane or filter. Suitable materials forcapturing genomic DNAs and viral nucleic acids include those marketed byQIAGEN under the name QIAmp, for analysis of blood, tissues, and viralRNAs; and suitable materials for capturing DNAs from plant cells andtissues include those marketed by QIAGEN under the name DNeasy.

Depending on the configuration of the device, the sample can be causedto flow through the enrichment channel by any of a number of differentmeans, and combinations of means. In some device configurations, it maybe sufficient to allow the sample to flow through the device as a resultof gravity forces on the sample; in some configurations, the device maybe spun about a selected axis to impose a centrifugal force in a desireddirection. In other embodiments, active pumping means may be employed tomove sample through the enrichment channel and enrichment means housedtherein. In other embodiments, magnetic forces may be applied to movethe sample or to capture or immobilize a paramagnetic bead-targetcomplex during wash and elution steps. In yet other embodiments of thesubject invention, electrodes may be employed to apply an electric fieldwhich causes fluid to move through the enrichment channel. An elutionliquid will then be caused to flow through the enrichment medium torelease the enriched sample fraction from the material and carry it tothe main electrophoretic flowpath. Generally, an applied electric fieldwill be employed to move the elution liquid through the enrichmentchannel.

Electrophoretic gel media may also be employed as enrichment means inthe subject applications. Gel media providing for a diversity ofdifferent sieving capabilities are known. By varying the pore size ofthe media, employing two or more gel media of different porosity, and/orproviding for a pore size gradient and selecting the appropriaterelationship between the enrichment channel and the main electrophoreticflowpath, one can ensure that only the analyte comprising fraction ofinterest of the initial sample enters the main electrophoretic flowpath.For example, one could have a device comprising an enrichment channelthat intersects the main electrophoretic channel, where the enrichmentchannel comprises, in the direction of sample flow, a stacking gel oflarge porosity and a second gel of fine porosity, where the boundarybetween the gels occurs in the intersection of the enrichment channeland the main electrophoretic flowpath. In this embodiment, after sampleis introduced into the stacking gel and an electric field applied to thegels in the enrichment channel, the sample components move through thestacking gel and condense into a narrow band at the gel interface in theintersection of the enrichment channel and main electrophoreticflowpath. A second electric field can then be applied to the mainelectrophoretic flowpath so that the narrow band of the enriched samplefraction moves into and through the main electrophoretic flowpath.Alternatively, the enrichment channel could comprise a gel of gradientporosity. In this embodiment, when the band(s) of interest reaches theintersection of the enrichment channel and electrophoretic flowpath, theband(s) of interest can then be moved into and along the mainelectrophoretic flowpath.

Enrichment media that can be particularly useful for enrichment and/orpurification of nucleic acids include sequence specific capture media aswell as generic capture media. Generic capture media include, forexample: ion exchange and silica resins or membranes whichnonspecifically bind nucleic acids, and which can be expected to retainsubstantially all the DNA in a sample; immobilized single-stranded DNAbinding protein (SSB Protein), which can be expected to bindsubstantially all single-stranded DNA in a sample; poly-dT modifiedbeads, which can be expected to bind substantially all the mRNA in asample. Sequence specific capture media include beads, membranes orsurfaces on which are immobilized any of a variety of capture moleculessuch as, for example: oligonucleotide probes, which can be expected tobind nucleic acids having complementary sequences in the sample;streptaviden, which can be expected to bind solution phase biotinylatedprobes which have hybridized with complementary sequences in the sample.Suitable beads for immobilization of capture molecules includechemically or physically crosslinked gels and porous or non-porousresins such as polymeric or silica-based resins.

Suitable capture media for proteins include the following. Suitablecapture media for proteins include: ion exchange resins, including anion(e.g., DEAE) and cation exchange; hydrophobic interaction compounds(e.g., C4, C8 and C18 compounds); sulfhydryls; heparins; inherentlyactive surfaces (e.g., plastics, nitrocellulose blotting papers);activated plastic surfaces; aromatic dyes such as Cibacron blue, Remazolorange, and Procion red. For carbohydrate moieties of proteins, lectins,immobilized hydrophobic octyl and phenylalkane derivatives can besuitable. For enzymes, analogs of a specific enzyme substrate-producttransition-state intermediate can be suitable; for kinases, calmodulincan be suitable. Suitable capture media for receptors include receptorligand affinity compounds.

As mentioned above, the enrichment channel will comprise at least oneinlet and at least one outlet. Of course, where there is a single inlet,the inlet must serve to admit sample to the enrichment channel at anenrichment phase of the process, and to admit an elution medium duringan elution phase of the process. And where there is a single outlet, theoutlet must serve to discharge the portion of the sample that has beendepleted of the fraction retained by the enrichment media, and to passto the main electrophoretic microchannel the enriched fraction duringthe elution phase. Depending on the particular enrichment means housedin the enrichment channel, as well as the particular deviceconfiguration, the enrichment channel may have more than one fluidinlet, serving as, e.g., sample inlet and elution buffer inlet; or theenrichment channel may have more than one outlet, serving as, e.g.,waste outlet and enriched fraction fluid outlet. Where the enrichmentchannel is in direct fluid communication with the main electrophoreticchannel, i.e., the enrichment channel and main electrophoretic flowpathare joined so that fluid flows from the enrichment channel immediatelyinto the main electrophoretic flowpath, the enrichment channel willcomprise, in addition to the waste outlet, an enriched fraction fluidoutlet through which the enriched fraction of the sample flows into themain electrophoretic flowpath. When convenient, e.g., for theintroduction of wash and/or elution solvent into the enrichment channel,one or more additional fluid inlets may be provided to conduct suchsolvents into the enrichment channel from fluid reservoirs. To controlbulk fluid flow through the enrichment channel, e.g., to prevent wastesample from flowing into the main electrophoretic flowpath, fluidcontrol means, e.g., valves, membranes, etc., may be associated witheach of the inlets and outlets. Where desirable for moving fluid andentities through the enrichment channel, e.g., sample, elution buffer,reagents, reactants, wash or rinse solutions, etc., electrodes may beprovided capable of applying an electric field to the material and fluidpresent in the enrichment channel.

The next component of the subject devices is the main electrophoreticflowpath. The main electrophoretic flowpath may have a variety ofconfigurations, including tube-like, trench-like or other convenientconfiguration, where the cross-sectional shape of the flowpath may becircular, ellipsoid, square, rectangular, triangular and the like sothat it forms a microchannel on the surface of the planar substrate inwhich it is present. The microchannel will have cross-sectional areawhich provides for capillary fluid flow through the microchannel, whereat least one of the cross-sectional dimensions, e.g., width, height,diameter, will be at least about 1 mm, usually at least about 10 mm, butwill not exceed about 200 mm, and will usually not exceed about 100 mm.Depending on the particular nature of the integrated device, the mainelectrophoretic flowpath may be straight, curved or another convenientconfiguration on the surface of the planar substrate.

The main electrophoretic flowpath, as well as any additionalelectrophoretic flowpaths, will have associated with it at least onepair of electrodes for applying an electric field to the medium presentin the flowpath. Where a single pair of electrodes is employed,typically one. member of the pair will be present at each end of thepathway. Where convenient, a plurality of electrodes may be associatedwith the electrophoretic flowpath, as described in U.S. Pat. No.5,126,022, the disclosure of which is herein incorporated by reference,where the plurality of electrodes can provide for precise movement ofentities along the electrophoretic flowpath. The electrodes employed inthe subject device may be any convenient type capable of applying anappropriate electric field to the medium present in the electrophoreticflowpath with which they are associated.

Critical to the subject invention is that the enrichment channel and themain electrophoretic flowpath are positioned in the device so thatsubstantially only the enriched fraction of the sample flows through themain electrophoretic flowpath. To this end, the device will furthercomprise a discharge outlet for discharging a portion of sample otherthan the enriched fraction, e.g., the waste portion, away from the mainelectrophoretic flowpath. Thus, where the enrichment channel is indirect fluid communication with the main electrophoretic flowpath, thewaste fluid flowpath through the enrichment channel will be in anintersecting relationship with the main electrophoretic flowpath. Inother embodiments of the subject invention where the enrichment channeland main electrophoretic flowpath are connected by a secondelectrophoretic flowpath so that they are in indirect fluidcommunication, the waste flowpath through the enrichment channel doesnot necessarily have to be in an intersecting relationship with the mainelectrophoretic flowpath; the waste flowpath and main electrophoreticflowpath could be parallel to one another.

The subject devices will also comprise a means for transferring theenriched fraction from the enrichment channel to the mainelectrophoretic flowpath. Depending on the particular deviceconfiguration, the enriched fraction transfer means can be an enrichedfraction fluid outlet, a secondary electrophoretic pathway, or othersuitable transfer means. By having a second electrophoretic flowpath inaddition to the main electrophoretic flowpath, the possibility exists toemploy the second electrophoretic flowpath as a conduit for the enrichedsample fraction from the enrichment channel to the main electrophoreticflowpath. In those embodiments where the waste outlet is the sole fluidoutlet, the presence of a secondary electrophoretic flowpath will beessential, such that the enrichment channel and the main electrophoreticflowpath are in indirect fluid communication.

In addition to the main and any secondary electrophoretic flowpathserving as an enriched sample transfer means, the subject devices mayfurther comprise one or more additional electrophoretic flowpaths, whichmay or may not be of capillary dimension and may serve a variety ofpurposes. With devices comprising a plurality of electrophoreticflowpaths, a variety of configurations are possible, such as a branchedconfiguration in which a plurality of electrophoretic flowpaths are influid communication with the main electrophoretic flowpath. See U.S.Pat. No. 5,126,022, the disclosure of which is herein incorporated byreference.

The main electrophoretic flowpath and/or any secondary electrophoreticflowpaths present in the device may optionally comprise, and usuallywill comprise, fluid reservoirs at one or both termini, i.e., eitherend, of the flowpaths. Where reservoirs are provided, they may serve avariety of purposes, such as a means for introducing buffer, elutionsolvent, reagent, rinse and wash solutions, and the like into the mainelectrophoretic flowpath, receiving waste fluid from the electrophoreticflowpath, and the like.

Another optional component that may be present in the subject devices isa waste fluid reservoir for receiving and storing the waste portion ofthe initial sample volume from the enrichment channel, where the wastereservoir will be in fluid communication with the discharge outlet.Depending on the particular device configuration, the discharge outletmay be the same as, or distinct from, the waste outlet, and may openinto a waste reservoir or provide an outlet from the device. The wastereservoir may be present in the device as a channel, compartment, orother convenient configuration which does not interfere with the othercomponents of the device.

The subject device may also optionally comprise an interface means forassisting in the introduction of sample into the sample preparationmeans. For example, where the sample is to be introduced by syringe intothe device, the device may comprise a syringe interface which serves asa guide for the syringe needle into the device, as a seal, and the like.

Depending on the particular configuration and the nature of thematerials from which the device is fabricated, at least in associationwith the main electrophoretic flowpath will be a detection region fordetecting the presence of a particular species in the medium containedin the electrophoretic flowpath. At least one region of the mainelectrophoretic flowpath in the detection region will be fabricated froma material that is optically transparent, generally allowing light ofwavelengths ranging from 180 to 1500 nm, usually 220 to 800 nm, moreusually 250 to 800 nm, to have low transmission losses. Suitablematerials include fused silica, plastics, quartz glass, and the like.

The integrated device may have any convenient configuration capable ofcomprising the enrichment channel and main electrophoretic flowpath, aswell as any additional components. Because the devices are microchannelelectrophoretic devices, the electrophoretic flowpaths will be presenton the surface of a planar substrate, where the substrate will usually,though not necessarily, be covered with a planar cover plate to seal themicrochannels present on the surface from the environment. Generally,the devices will be small, having a longest dimension in the surfaceplane of no more than about 200 mm, usually no more than about 100 mm sothat the devices are readily handled and manipulated. As discussedabove, the devices may have a variety of configurations, includingparallelepiped, e.g., credit card or chip like, disk like, syringe likeor any other compact, convenient configuration.

The subject devices may be fabricated from a wide variety of materials,including glass, fused silica, acrylics, thermoplastics, and the like.The various components of the integrated device may be fabricated fromthe same or different materials, depending on the particular use of thedevice, the economic concerns, solvent compatibility, optical clarity,color, mechanical strength, and the like. For example, both the planarsubstrate comprising the microchannel electrophoretic flowpaths and thecover plate may be fabricated from the same material, e.g.,polymethylmethacrylate (PMMA), or different materials, e.g., a substrateof PMMA and a cover plate of glass. For applications where it is desiredto have a disposable integrated device, due to ease of manufacture andcost of materials, the device will typically be fabricated from aplastic. For ease of detection and fabrication, the entire device may befabricated from a plastic material that is optically transparent, asthat term is defined above. Also of interest in certain applications areplastics having low surface charge under conditions of electrophoresis.Particular plastics finding use include polymethylmethacrylate,polycarbonate, polyethylene terepthalate, polystyrene or styrenecopolymers, and the like.

The devices may be fabricated using any convenient means, includingconventional molding and casting techniques. For example, with devicesprepared from a plastic material, a silica mold master which is anegative for the channel structure in the planar substrate of the devicecan be prepared by etching or laser micromachining. In addition tohaving a raised ridge which will form the channel in the substrate, thesilica mold may have a raised area which will provide for a cavity intothe planar substrate for housing of the enrichment channel. Next, apolymer precursor formulation can be thermally cured or photopolymerizedbetween the silica master and support planar plate, such as a glassplate. Where convenient, the procedures described in U.S. Pat. No.5,110,514, the disclosure of which is herein incorporated by reference,may be employed. After the planar substrate has been fabricated, theenrichment channel may be placed into the cavity in the planar substrateand electrodes introduced where desired. Finally, a cover plate may beplaced over, and sealed to, the surface of the substrate, therebyforming an integrated device. The cover plate may be sealed to thesubstrate using any convenient means, including ultrasonic welding,adhesives, etc.

Generally, prior to using the subject device, a suitable first orelectrophoretic medium will be introduced into the electrophoreticflowpaths or microchannels of the device, where the first medium will bedifferent from the enrichment medium present in the enrichment channel.Electrophoretic media is used herein to refer to any medium to which anelectric field is applied to move species through the medium. Theelectrophoretic media can be conveniently introduced through thereservoirs present at the termini of the electrophoretic flowpaths ordirectly into the channels or chambers of the electrophoretic flowpathsprior to sealing of the cover plate to the substrate. Any convenientelectrophoretic medium may be employed. Electrophoretic media suitablefor use, depending on the particular application, include buffers,crosslinked and uncrosslinked polymeric media, organic solvents,detergents, and the like, as disclosed in Barron & Blanch, “DNASeparations by Slab Gel and Capillary Electrophoresis: Theory andPractice,” Separation and Purification Methods (1995) 24:1-118, as wellas in U.S. patent applications Ser. Nos. 08/636,599 and 08/589,150 andU.S. Pat. No. 5,569,364, the disclosures of which are hereinincorporated by reference. Of particular interest as electrophoreticmedia are cellulose derivatives, polyacrylamides, polyvinyl alcohols,polyethylene oxides, and the like.

The subject invention will now be further described in terms of thefigures. FIG. 1 provides a diagrammatic view of an enrichment channelwhich may find use in the devices of the subject invention. Enrichmentchannel 10 comprises side walls 1 which enclose reverse phase C18material 2. Channel 10 further comprise fluid inlets 7 and 4 and fluidoutlets 5 and 6. For controlling fluid flow through the channel inletsand outlets, valves 8, 9 and 11 are provided. Glass frits 3 allow forfluid flow through inlet 4 and outlet 5 but restrain reverse phasematerial 2 in the channel. In using this enrichment channel, sample isintroduced through sample inlet 7 in the direction of flowpath 12. Assample moves through channel 10, the analyte comprising fraction isretained on reverse phase material 2 while the remaining waste fractionof the sample flows out waste outlet 6 along flowpath 13. Valves 8 and 9are closed to prevent sample from flowing or “bleeding” out inlet 4 oroutlet 5. After the sample has flowed through channel 10, valve 11 isshut and valves 8 and 9 are opened. Elution buffer is then introducedinto channel 10 through glass frit 3 and inlet 4 in the direction offlowpath 14. As elution buffer moves through material 2, the retainedfraction of the sample is released and carried with the elution bufferout enriched fraction outlet 5 through frit 3 along flowpath 15.

In FIG. 2, the same enrichment channel as shown in FIG. 1 is depictedwith the exception that reverse phase material 2 is replaced by anetwork of crosslinked glass filaments 16 to which binding pair memberis covalently bound.

FIG. 3A provides a diagrammatic top view of a credit card shaped(parallelepiped) device according to the subject invention. Device 30comprises main electrophoretic flowpath 31 having reservoir 32 at afirst end and reservoir 33 at a second end. In direct fluidcommunication with main electrophoretic flowpath 31 is enrichmentchannel 34 (seen from overhead). Electrodes 35 and 36 are provided forapplying an electric field to the medium present in electrophoreticflowpath 31. Detection region 37 is positioned over electrophoreticflowpath 31 for viewing analyte present in the medium comprised in theflowpath. A detection region can also be provided over the enrichmentchannel 34. Although the device shown in FIG. 3A comprises a singleenrichment channel, additional enrichment channels could be provided inthe flowpath, including in the detection region.

FIG. 3B provides a diagrammatic side view of the device depicted in FIG.3A. In using this embodiment of the subject invention, sample isintroduced through syringe interface 38 into enrichment channel 34,where the analyte comprising fraction of the sample is retained as thewaste fraction flows out of the enrichment channel 34 through dischargeoutlet 39 and out of the device. Elution buffer is then introduced intoreservoir 32 through port 40. An electric field is then applied betweenelectrodes 35 and 36 causing elution buffer to migrate from reservoir 32through enrichment channel 34 and along electrophoretic flowpath 31 toreservoir 33. As the elution buffer moves through enrichment channel 34,it releases the retained analyte comprising fraction of the initialsample volume and carries it into electrophoretic flowpath 31.

FIG. 4 shows a diagrammatic view of an embodiment of the subjectinvention in which the enrichment channel 62 is separated from mainelectrophoretic flowpath 52 by secondary electrophoretic flowpath 55.With device 50, sample is introduced into enrichment channel 62 throughsyringe interface 66. As sample flows through enrichment channel 62,waste sample flows through discharge outlet 64 into waste reservoir 63.An electric field is then applied between electrodes 61 and 60 causingelution buffer present in reservoir 57 to move through enrichmentchannel 62, resulting in the release of analyte. Analyte is then carriedalong secondary electrophoretic flowpath 55 along with the elutionbuffer. When analyte reaches intersection 51, the electric field betweenelectrodes 60 and 61 is replaced by an electric field between electrodes59 and 58. In this and other analogous embodiments of the subjectinvention, the time at which analyte reaches intersection 51 may bedetermined by detecting the presence of analyte in the intersection orby empirically determining the time at which the analyte should reachthe intersection, based on the particular nature of the analyte, themedium in the flowpath, the strength of the electric field, and thelike. Following application of the electric field between electrodes 59and 58, which are placed in reservoirs 54 and 53 respectively, theanalyte moves from intersection 51 along electrophoretic flowpath 52towards reservoir 53 and through detection region 65.

FIG. 5 provides a diagrammatic top view of yet another embodiment of thesubject invention in which the enrichment channel comprises a singlefluid inlet and outlet. Device 70 comprises main electrophoreticflowpath 71 in intersecting relationship with secondary electrophoreticflowpath 73. Upstream from the intersection 82 along secondaryelectrophoretic flowpath 73 is enrichment channel 72. In using thisembodiment, sample is introduced through syringe interface 80 intoenrichment channel 72, whereby the analyte comprising fraction of thesample is reversibly bound to the material present in the enrichmentchannel. An electric field is then applied between electrodes 81 and 79which moves the non-reversibly bound or waste fraction of the sample outof the enrichment channel 72, along secondary electrophoretic flowpath73, past intersection 82, and out discharge outlet 84 into wastereservoir 78. An elution buffer is then introduced into enrichmentchannel 72 through syringe interface 80 and an electric field appliedbetween electrodes 81 and 79, causing elution buffer to flow throughenrichment channel 72 into secondary flow electrophoretic flowpath 73,carrying analyte along with it. When analyte reaches intersection 82,the electric field between electrodes 79 and 81 is replaced by anelectric field between electrodes 76 and 77, which causes analyte tomove along main electrophoretic flowpath 71 and towards reservoir 74through detection region 99.

The device shown diagrammatically in FIG. 6 comprises an enrichmentchannel having an electrophoretic enrichment means, instead of thechromatographic enrichment means of the devices of FIGS. 1 to 5. Indevice 90, sample is introduced into reservoir 96 and an electric fieldis applied between electrodes 87 and 88, causing the sample to migratetowards reservoir 98. As the sample migrates towards reservoir 98 itenters stacking gel 93 having a relatively large pore size and travelstowards secondary gel 92 of relatively fine pore size. At interface 94,the sample components are compressed into a narrow band. At this point,the electric field between electrodes 87 and 88 is replaced by anelectric field between electrodes 89 and 90, which causes the narrowband of sample components at interface 93 to migrate into mainelectrophoretic flowpath 95, past detection region 91 and towardsreservoir 85. In device 90, instead of the stacking gel configuration,one could provide for a molecular size membrane at the region ofinterface 93, which can provide for selective passage of samplecomponents below a threshold mass and retention at the membrane surfaceof components in excess of the threshold mass. In yet anothermodification of the device shown in FIG. 6, present at the location ofinterface 93 could be an electrode by which an appropriate electricpotential could be applied to maintain a sample component of interest inthe region of 93, thereby providing for component concentration in theregion of 93. For example, for an anionic analyte of interest, uponintroduction of sample into reservoir 96 and application of an electricfield between 93 and 87, in which 93 is the positive electrode and 87the ground, the anionic will migrate towards and concentrate in theregion of 93. After the analyte has concentrated in the region ofelectrode 93, an electric field can then be applied between 89 and 90causing the anionic analyte to migrate towards reservoir 85.

FIG. 7 provides a top diagrammatic view of a disk shaped embodiment ofthe subject device, as opposed to the credit card shaped embodiments ofFIGS. 3 to 6. In device 100, sample is first introduced into enrichmentchannel 102. An electric field is then applied between electrodes 108and 109, moving elution buffer 103 through enrichment channel 102,whereby analyte retained in the enrichment channel 102 is released andcarried with the elution buffer to intersection 114. The electric fieldbetween 108 and 109 is then replaced with an electric field between 110and 111, causing analyte to move from intersection 114 along mainelectrophoretic flowpath 112, past detection region 113 and towardsreservoir 107.

Other embodiments may be understood by reference to the flow diagrams inFIGS. 8 through 19, some of which correspond to embodiments shown in thesketches of FIGS. 1 through 7. Referring, for example, to FIG. 8, thereis shown a flow diagram of an enrichment channel as shown in FIG. 1 orFIG. 2, with corresponding identification numbers. Accordingly, asdescribed with reference to FIGS. 1 and 2, sample enters enrichmentchannel 10 through sample inlet 7 by way of flowpath 12. As the samplemoves through enrichment channel 10 the fraction containing the fractionof interest is retained on an enrichment medium, which may be, forexample, a reverse phase C18 material (as described with reference toFIG. 1) or binding pair members covalently bound to a network of glassfilaments (as described with reference to FIG. 2), while the remainingwaste fraction flows out through waste outlet 6 along flowpath 13. Aftera suitable quantity of sample has flowed through enrichment channel 10,flow through inlet 7 and outlet 6 is halted, and elution buffer entersenrichment channel 10 through inlet 4 by way of flowpath 14. Withinenrichment channel 10 the retained fraction of interest is released intothe elution buffer passing over the enrichment medium, and passes outthrough enriched fraction outlet 5 by way of flowpath 15.

And referring to FIG. 9, there is shown a flow diagram of the embodimentof a device 30 as sketched in two views in FIGS. 3A, 3B and describedwith reference thereto. In the flow diagrams, the enrichment channel (34in FIGS. 3A, 3B, 9) is represented by a square; the various reservoirs(e.g., 32, 33 in FIGS. 3A, 3B, 9) are represented by small circles atthe ends of the flowpaths (channels), which are represented by lines(e.g., main electrophoretic flowpath 31 in

FIGS. 3A, 3B, 9); electrodes (35, 36 in FIGS. 3A, 3B, 9) are representedby hairlines running to the centers of the reservoir circles; aninterface for syringe injection (where one may be present; e.g., 38 inFIGS. 3B, 9) is represented by a trapezoid at the end of the sampleinput flowpath; and the detection region (37 in FIGS. 3A, 3B, 9) isrepresented by a heavy arrow touching the main electrophoretic channel.Similarly, in FIG. 12, there is shown a flow diagram of the embodimentof a device 90 as sketched in FIG. 6 and described above with referencethereto. In this embodiment, the enrichment channel (120 in FIG. 12)works by electrophoretic enrichment, which results in accumulation ofthe fraction of interest at the point where the enrichment channel 120is intersected by the main electrophoretic channel 95. Movement ofsample material through the enrichment channel can be accomplished byapplication of an electrical potential difference between electrodes 87,88; and elution of the fraction of interest from the enrichment channelthrough the main electrophoretic channel and to the detection region 91can be accomplished by application of an electrical potential differencebetween electrodes 89, 90. As described above with reference to FIG. 6,the accumulation point can be an interface 94 between a stacking gel 93and a secondary gel 92; and in a further modification, a suitableelectrical potential can be applied at an electrode (121 in FIG. 12) atthe site of the interface 93 to provide for component concentration inthat region of the enrichment channel.

FIG. 10 is a flow diagram of the embodiment of a device 50 in which theenrichment channel 62 is separated from main electrophoretic flowpath 52by secondary electrophoretic flowpath 55, as sketched in FIG. 4 anddescribed above with reference thereto. Similarly, FIG. 13 is a flowdiagram of the disc-shaped embodiment of a device 100 as sketched inFIG. 7 and described with reference thereto. FIG. 13 shows the sampleinput flowpath by which the sample is introduced from the syringeinterface 66 into the enrichment channel 102, and the discharge outlet64 by which waste passes out to waste reservoir 63 while the fraction ofinterest is retained on the retention medium in the enrichment channel.These features are not shown in the top views of FIG. 7 or FIG. 4.

In FIG. 11 there is shown a flow diagram of a device 70, in which thereis only one fluid inlet into, and one fluid outlet out from, theenrichment channel 72, as sketched in FIG. 5 and described withreference thereto. During sample injection by way of the syringeinterface the fluid inlet 116 serves as a sample inlet and the fluidoutlet 118 serves as a waste outlet. While the fraction of interest isretained by the retention medium in the enrichment channel, the wastefraction flows downstream through the secondary electrophoretic flowpath73, across the intersection 82 of the secondary electrophoretic flowpathwith the main electrophoretic flowpath 71, and into discharge outlet 84,which directs the waste away from the mail electrophoretic flowpath 71toward waste reservoir 78. During elution, elution buffer is injected byway of the syringe interface; fluid inlet 116 serves as an elutionbuffer inlet and the fluid outlet 118 serves as an enriched fractionoutlet to the secondary electrophoretic channel. The fraction ofinterest moves into the elution buffer in which it is drivenelectrokinetically in an electric field produced by applying a voltageacross electrodes 79, 81 to the intersection of the secondaryelectrophoretic channel and the main electrophoretic channel. Once thefraction of interest has reached the intersection, a voltage is appliedacross electrodes 76, 77 to draw the analyte or analytes in the fractionof interest into and along the main electrophoretic flowpath to thedetection zone 99.

As noted with reference to FIG. 5, the waste fraction (material notbound to the enrichment medium) can be washed out of the enrichmentchannel and away from the main electrophoretic pathway by application ofan electric field between electrodes upstream from the enrichmentchannel and downstream from the discharge outlet. That is, prior tointroducing the elution buffer to the enrichment channel, a liquid washmedium is passed over the enrichment medium and out through thedischarge outlet, carrying away waste fraction components. Any of avariety of materials can be suitable as a wash medium, so long as thewash medium does not substantially elute the fraction of interest fromthe enrichment medium. Moreover, the wash medium can be chosen tofacilitate a selective release or removal, prior to elution, ofundesired components that may be bound to or otherwise associated withthe enrichment medium. For example, where the components of interest areDNA fragments, the wash medium may contain enzymes that selectivelydegrade proteins or polypeptides or that selectively degrade RNAs,facilitating the removal of these contaminants away from the fraction ofinterest prior to elution. Or, for example, where the components ofinterest are proteins, the wash medium may contain DNAses and RNAses.

Sequential movement of the various liquids into and through theenrichment channel can be readily controlled by providing a reservoirand a flowpath to the upstream part of the enrichment channel for eachsuch liquid. As illustrated in the flow diagram of FIG. 14, for example,an input 212 to enrichment channel 210 is fed by a sample supplyflowpath 220 running from a sample reservoir 218, by a wash mediumflowpath 218 running from a wash medium reservoir 217, and by an elutionmedium flowpath 216 running from an elution medium reservoir 215.Movement of these materials can be selectively controlled by applicationof electrical potentials across electrodes (not shown the Fig.) at therespective reservoirs and at suitable points (as described herein forvarious configurations) downstream from enrichment channel output 214.Suitable wash media for proteins include, for example, pH-adjustedbuffers and organic solvents; and washing can be effected by, forexample, adjusting ionic strength or temperature of the wash medium.

Other materials may be introduced to the input flowpath as well, and,particularly, one or more reagent streams can be provided forpretreatment of the sample itself prior to moving it onto the enrichmentchannel. A crude sample of body fluid (blood, lymphatic fluid, amnioticfluid, cerebrospinal fluid, or urine, for example) can be pretreated bycombining the sample with a reagent in the sample flowpath. For example,DNA may be released from cells in a crude sample of whole blood byadmixture of a reagent containing an enzyme or a detergent.

Other flowpath configurations downstream from the enrichment channel canbe employed, and certain of these may provide some advantages forparticular kinds of downstream treatment or analysis of the componentsof the fraction of interest. In FIG. 15, for example, the secondaryelectrophoretic flowpath does not cross the main electrophoreticflowpath; rather, main electrophoretic flowpath 238 joins secondaryelectrophoretic flowpath 236 at a T intersection (compare, FIG. 12). Inthis configuration, the upstream arm of the main electrophoreticflowpath runs in the same channel as the secondary electrophoreticflowpath 236. As in other configurations, described herein, sampleenters the enrichment channel 230 by way of sample flowpath 234 fromsample reservoir 233; and during the enrichment stage the waste fluidpasses out from enrichment channel 230 by way of secondaryelectrophoretic flowpath 236, then past T intersection 237 and awaythrough discharge outlet 240 to waste reservoir 241. Once the enrichmentstage is complete, a wash medium may be passed through the enrichmentchannel and also out through the discharge outlet. The wash medium maybe introduced by way of the sample supply flowpath or, optionally, froma separate wash medium flowpath as described above with reference toFIG. 14. Movement of the sample and the wash medium can be accomplishedby application of an electric field across electrodes (not shown in theFig.) at waste reservoir 241 and, respectively, sample reservoir 233(and, optionally, a wash reservoir). Then, an elution medium can bemoved from an elution buffer reservoir 231 by way of elution bufferpathway 235 into and through enrichment channel 230, through secondaryelectrophoresis pathway 236. Media downstream from the eluting fractioncomponents can be directed away from main electrophoretic flowpath 238and out by way of waste discharge flowpath 240, until the mostdownstream component of interest has reached the intersection 237. Thenan electrical potential can be applied at reservoir 239 to draw thecomponents from secondary electrophoretic flowpath 236 throughintersection 237 and within main electrophoretic flowpath 238 toward andthrough detection region 242.

An intersection of the main and secondary electrophoretic flowpaths atan “injection cross”, as shown for example in FIGS. 5, 12, can beadvantageous where precise metering of the sample plug is desired, asfor example, where the main electrophoretic flowpath is used forelectrophoretic separation. Such an injection cross can provide forinjection from the intersection of a geometrically defined plug ofsample components from the fraction of interest.

On the other hand, where precise control of a sample plug is notdesirable, and particularly where it is desirable to move the entireeluted sample through the main electrophoretic path way, a Tintersection can be preferred. Such a configuration may be advantageouswhere, for example, the components are analyzed by passing substantiallythe entire eluted fraction through an array of affinity zones downstreamfrom the intersection.

By way of example, FIG. 16 is a diagram showing the flow in aconfiguration having a serial array of affinity zones 244, 246, 248,250. Each affinity zone is provided with an enrichment medium that has aspecific affinity for a selected component of the fraction of interest.For example, the fraction of interest may consist of DNA in a crude celllysate, wherein the lysate may have been formed upstream from enrichmentchannel 230 and concentrated and/or purified in enrichment channel 230,so that the eluted fraction that passes into main electrophoreticflowpath 238 consists principally of a complex mixture of DNA fragmentsof different lengths and base composition. Each hybridization zone isitself an enrichment channel, in which the enrichment medium includes animmobilized oligonucleotide probe having a sequence complementary to asequence in a target DNA. As the eluted fraction passes serially throughthe affinity zones 244, 246, 248, 250, any target DNA present in thefraction that is complementary to the probe in one of the affinity zoneswill become bound in that affinity zone. The affinity zones are providedwith detectors 243, 245, 247, 249, configured to detect and, optionally,to quantify, a signal (such as fluorescence orelectrochemilluminescence) from components of interest bound in theaffinity zones. Any form of biomolecular recognition may be employed asa capture principle in the affinity zones, as the skilled artisan willappreciate. Useful types of affinity include antibody-antigeninteractions; binding of poly-dT with adenylated RNA; oligonucleotideprobes for RNA, DNA, PNA; streptavidin-biotin binding; protein-DNAinteractions, such as DNA-binding protein G or protein A; and moleculeshaving group specific affinities, such as arginine, benzamidine,heparin, and lectins. Other examples will be apparent to the skilledartisan.

Accordingly, for example, the capture principle may includereceptor-ligand binding, antibody-antigen binding, etc., and thus themethods and devices according to the invention can be useful forcarrying out immunoassays, receptor binding assays, and the like, aswell as for nucleic acid hybridization assays.

Alternatively, as mentioned above, the main electrophoretic flowpath canbe branched downstream from the intersection with the secondaryelectrophoretic flowpath, providing a parallel array of mainelectrophoretic flowpaths, as shown by way of example in FIG. 17.Electrophoretic flowpath 238 is shown as twice bifurcated, so that fourmain electrophoretic flowpath branches run downstream to theirrespective waste reservoirs 262, 264, 266, 268. The branches areprovided in this example with affinity zones 254, 256, 258, 260, withdetectors 253, 255, 257, 259. Pertinent properties of the milieu (suchas, e.g., temperature, pH, buffer conditions, and the like) canadvantageously be controlled in each flowpath branch independently ofthe others, as is shown in more detail with reference to FIG. 22, below.

Where the affinity zones are arranged in parallel, as for example inFIG. 17, each affinity zone receives an aliquot of the entire samplethat is delivered to the main electrophoresis channel. In thisembodiment, sample components that can be captured by two or more of theaffinity media will appear in the respective two or more affinity zones.For example, a nucleic acid fragment that contains either one or both oftwo sequences complementary to two of the probe sequences will, in theparallel arrangement, be captured in the two affinity zones containingthose two probes. On the other hand, where the affinity zones areserially arrayed, as for example in FIG. 16, each downstream affinityzone is reached only by sample components not captured by an affinityzone upstream from it. Here, for example, a nucleic acid fragment thatcontains both of two sequences complementary to probe sequences in twoof the affinity zones will be captured only in the more upstream of thetwo affinity zones. This arrangement may be advantageous where it isdesirable to identify sample components that contain one but not anothermoiety or sequence.

And alternatively, as noted above, a plurality of main electrophoreticflowpaths may be provided for treatment of the enriched eluted sample.As shown by way of example in FIG. 18, the main electrophoreticflowpaths 270 may carry eluted sample fraction from the secondaryelectrophoretic flowpath 236 through a series of intersections 272. Eachmain electrophoretic flowpath 270 is provided with reservoirs upstream(274) and downstream (276) and each is provided with a detector 278.This configuration may be employed to run a set of tests or assays ormeasurements on aliquots of a single enriched sample fraction, and willbe particularly useful where, as noted above, precise metering of thequantity of analyte is desirable. As will be appreciated, each of themain electrophoretic flowpaths 270 can be provided with an affinity zoneor with an array of affinity zones (not shown in FIG. 18) as describedabove with reference to FIGS. 16, 17.

Or, as shown by way of example in FIG. 19, a plurality of enrichmentchannels 280 can receive sample from a branched sample supply manifold281. Each enrichment channel 280 can during the elution stage deliver anenriched fraction to an intersection 288 with a main electrophoreticflowpath 284. During the enrichment stage (and optionally during a washstage) waste fraction is carried away from the intersections 288 by wayof a branched discharge manifold 283 and out through discharge outlet240 to waste 241. Such an arrangement can be used to particularadvantage, for example, where the fraction of interest is a mixture ofDNAs, and where it is desirable to obtain both sequence information andsize information for the DNAs. The configuration of FIG. 19 can be used,for example, for a flow-through analysis analogous to a Southern blotanalysis. In the conventional Southern blot analysis, DNA fragments arefirst separated on a gel, and then transferred to a membrane on whichprobes are allowed to bind complementary fragments. The Southern blotanalysis is practiced mainly as a manual bench-top procedure, and ishighly labor-intensive, taking several days to complete. Theflow-through analysis, according to the invention, can be substantiallyautomated, and the analysis can be completed much more rapidly.

In the flow-through analysis, each but one of the enrichment channels isprovided with a sequence-specific capture medium, such as asequence-specific immobilized oligonucleotide probe, and the last one ofthe enrichment channels is provided with a generic capture medium whichbinds all DNA fragments in the sample. These different enrichedfractions are delivered to the intersections 288 during the elutionstage, and then they are moved electrophoretically in the respectivemain electrophoretic flowpaths 284, each provided with a detector 286.The enriched fraction from the enrichment channel containing a genericcapture medium contains a mixture of all sizes of DNAs from the sample,having a range of electrophoretic mobilities, passing the detectorsequentially, and resulting in a series of signal peaks. The enrichedfraction from each of the other enrichment channels contains only DNAscomplementary to the specific capture medium in its respectiveenrichment channel.

The use of affinity binding agents on particulate supports can, incertain configurations of flowpaths, provide for highly efficientseparation of a selected subset of biological entities from among two ormore subsets in a mixed population of biological entities, where eachsubset has a characteristic determinant. For example, several enrichmentchannels, or affinity zones, in each of which is held a capture agentcapable of selectively binding a determinant on a subset of biologicalentities, can be arranged in parallel. The capture agents include afirst capture agent comprising a receptor which specifically binds,either directly or indirectly, to the characteristic determinant of thefirst subset, and at least a second capture agent comprising a receptorwhich specifically binds, either directly or indirectly, to thecharacteristic determinant of at least one other subset. The subset towhich each capture agent binds is the target subset of each captureagent. A sample of the mixed population of biological entities iscontacted with the plurality of capture agents, under conditionsfavoring specific binding of the receptor of the first capture agent tothe first subset, and of the receptor of the second capture agent to atleast one other subset, where at least one of the capture agents isdissociably bound to its respective subset.

The bound subsets are next separated from the sample and from any subsetof the population of biological entities that is not bound to a captureagent. One of the dissociably bound subsets is thereafter dissociatedfrom the capture agent to which it is bound, and is thereafter isolated.The isolated, selected subset is normally recovered for furtherprocessing, which may include analysis and/or propagation.

These dissociation and isolation steps as described above may berepeated to yield a second or third selected subset, and so on, ifdesired, provided that dissociation of the one capture agent from itstarget subset does not result in dissociation of another capture agentfrom its selected target subset.

According to the device and method of this embodiment of the invention,operating parameters and device configuration enable successfulperformance of biological and other separations not heretoforeattainable. In conventional affinity separations, wherein a ligand isattached directly to a stationary solid support, such as in affinitychromatography, capture and separation of the target substance aresimultaneous events. For separations using a particulate magneticcapture agent, as in an embodiment of the present invention, these twoevents are separate. The bifurcation of these two events according to apreferred embodiment of this invention affords significant advantages.

In the method of the invention, affinity-binding reactions are coupledwith respective specific cleavage reaction. Thus, by creatingaffinity-binding/cleavage pairs, two distinct specificities for eachseparation procedure result. When it is desired to separate one or moreselected subset of biological entities from a mixed population of suchentities on a collection surface, this additional parameter allowspermutations of events, such that separations which were eitherdifficult or impossible can be carried out according to the inventionwith relative ease.

Prior to the invention, a notable obstacle to the use of particles forthe separation and subsequent release of distinct, selected subsets froma mixed population of biological entities has been that the biologicalentities must be collected in such a manner as to allow the selectedsubset to be removed from the mixed population without appreciablecontamination from non-selected substances. In the practice of thepresent invention, this difficulty is overcome in two ways. One is inthe design of the integrated microfluidic device configuration. By theuse of apparatus and methods described in the above-referenced U.S. Ser.Nos. 08/690,307 and 08/902,855, which are commonly owned with thepresent application, and which are incorporated by reference in thepresent application as if set forth herein in full, it is possible tocircumvent the contamination problem. For example, a multiple parallelmicrochannel configuration provides for highly efficient separations.The second way involves the high degree of control that is afforded overthe collection of the biological entities, such that after an individualaffinity bond between the biological entity and the solid support iscleaved, which may be either before or after resuspension of thecollected biological entities, a second collection of the particlesresults in segregation of the original mixed population with theexception of the subset of biological entities that was bound by thespecific receptor which was selectively released from its target subsetvia bond cleavage.

Unlike the methods described for example in U.S. Patent No. 5,646,001,which is incorporated by reference in the present application as if setforth herein in full, the present invention is not limited to theselected control and manipulation of the physiochemical environmentassociated with bond breaking and deposition of the captured biologicalsubstances. Instead, a multiplexed microfluidic configuration providesenormous flexibility in the design of integrated devices for theseparation of mixtures of biological components. Thus, a combination ofboth approaches may be utilized in cases when multiple subsets ofbiological entities are to be isolated from a mixed cell populationwhich vary greatly in frequencies.

Reference is now made to FIG. 27, showing a configuration of flow pathsin a microfluidic device according to the invention that can be used forseparation of a mixture of five different biological entities (here,different cell types presenting as determinants different cell surfacereceptors) into four separate subsets.

The separation device and method provide for efficient isolation of anyof a broad range of biological entities, which may be a components of atest sample or specimen capable of selective interaction with a receptoror other specific binding substance. The term “biological entity” asused herein refers to a wide variety of substance of biological originincluding cells, and cell components such as membranes, organelles,etc., microbes, viruses, as well as molecules (e.g., proteins) andmacromolecules (e.g., nucleic acids, including RNAs, DNAs and PNAs).

The biological entities of interest may be present in test samples orspecimens of a wide range of origins, including for example biologicalfluids or extracts, food samples, environmental samples, etc.

The term “determinant” is used here in a broad sense to denote anycharacteristic that identifies or determines the nature of an entity.When used in reference to any of the above-described biologicalentities, determinant means that portion of the biological entityinvolved in and responsible for selective binding to a specific bindingsubstance, the presence of which is required for selective binding tooccur.

The expression “specific binding substance” as used herein refers to anysubstance that electively recognizes and interacts with thecharacteristic determinant on a biological entity of interest, to thesubstantial exclusion of determinants present on biological entitiesthat are not of interest.

The capture agents used in the affinity binding separations include aspecific binding agent, or receptor, attached to a solid support. Thesolid support may be either stationary or mobile. Useful mobile solidphases include, for example, beads and particles. Particulate solidsupports are preferably made from magnetic material to facilitatecapture of the target subsets by application of a magnetic field.

In a microfluidic device configured generally as illustrated in FIG. 27,and described with reference thereto, a heterogeneous mixture ofbiological entities is separated into subpopulations as characterized bythe determinants of the constituents of the sample.

As employed for isolation and purification of a subset of two or moresubpopulations of cells in a mixed population in a sample, the method issimple, rapid and reliable. Antibodies specific to corresponding cellsurface antigens serve as capture reagents for isolating the specifictargets from complex mixtures. The mixed population of biologicalentities may also include, but is not limited to, whole cells presentingcell surface receptors, cell membranes bearing cell surface receptors,soluble receptors, enzymes, antibodies, and specific nucleic acidsequences. Thus, a wide variety of applications involving cell biology,molecular biology, tissue typing, and microbiology are thereforepossible.

The integrated device as shown in FIG. 27 includes duplicate flowpatterns configured in four parallel networks of microchannels (denotedA, B, C, and D) for illustration purposes. A highly multiplexedconfiguration comprising of many parallel networks (more than four) is,as will be appreciated, contemplated within the invention. Similar indesign to the flow configuration of FIG. 15, each microfluidic networkincludes a capture channel (or “enrichment zone”), having specificcapture reagents (in this case, immobilized antibodies), in fluidcommunication with two inlet and two outlet flowpaths. With referencenow to network A, the inlet and outlet flow paths join the capturechannel 541 at intersections 531 and 571, respectively. One inletflowpath is supplied by sample inlet reservoir 502, which serves as thecommon inlet for the entire device, and microchannels 504, 506, and 511.The other inlet flowpath, specific to network A, comprises elutionbuffer reservoir 501 and microchannel 521. One outlet flowpath comprisesof the common outlet reservoir 592 and microchannels 561, 594 and 596.The other outlet flowpath comprises the analysis channel 551, outletreservoir 591 and the detection zone 581.

The three stage cell isolation process, including affinity capture,release and detection, is initiated by injecting a complex mixture ofbiological cells into the multiplexed flow pattern as schematicallyillustrated in FIG. 27. Sample handling on the microfluidic device isachieved electrokinetically by controlling the electric potential acrossthe appropriate electrodes (not shown in FIG. 27) placed within theinlet and outlet reservoirs. Within the enrichment zones, cells arecaptured by means of antibodies immobilized to the surface of thechannels that recognize specific cell surface antigens. Alternatively,immunomagnetic beads may be employed for cell capture. In this case, theheterogeneous suspension of cells bind the target (e.g., antibodies tocell surface antigens) by specific absorption to the particular capturemoieties on the surface of the beads. Immobilization of the target-beadcomplex to the side of enrichment chambers can then be achievedmagnetically.

Using the device as illustrated in FIG. 27, a mixture of, e.g., sixdifferent cell types can be separated into four distinct subsets whenbound to capture agents including four antibodies having differentbinding specificities. In this example, antibodies to cell surfaceantigens denoted by A, B, C, and D are immobilized in channels 541, 543,545, and 547, respectively. As will be appreciated, each of theenrichment channels 541, 543, 545, and 547 has associated with it acorresponding set of intersecting inlet and outlet flowpaths andreservoirs, analysis channels and detection zones. Thus, cells denotedA, B, C, and D arising from their respective surface antigens arecaptured in the above-referenced channels within the microfluidicnetworks A, B, C and D. The remaining cells are passed through thedevice and collected in the common outlet reservoir 592. The remainingcells may then be used in various applications as described furtherbelow.

Upon completion of the capture step, a wash medium contained within washbuffer reservoirs (not shown in the FIG. 27) may be used to rinse theimmobilized cells. The isolated cells captured in their respectiveenrichment zones can next be released and then analyzed within thedetection zones 581, 583, 585, and 587 by electrokinetically pumpingelution buffer from reservoirs 501, 503, 505, and 507 to the outletreservoirs 591, 593, 595, and 597, respectively. Depending on thedemands of the analyses and the particular application, the detectionzones may simply be an optical detector, e.g., fluorescence detector orthe like, or it may represent a further flow configuration. Finally,this embodiment of the invention affords an advantageous means forisolating and enriching the target biomolecules from a sample mixture.

Although the affinity-capture microchannels shown in FIG. 27 are in aparallel configuration, a single heterogeneous enrichment zone mayalternatively be employed with a plurality of receptors (e.g., in thiscase, antibodies specific to the cell surface antigens) immobilized tothe affinity channel. Heterogeneous capture and release methods aredescribed in, e.g., U.S. Pat. No. 5,646,001 to Terstappen et al., whichis incorporated herein by reference in its entirety. However, anadvantage of the parallel approach is that separate homogeneous capturezones minimize the physical impact on the biological entities. This isespecially important when working with whole cells, which can be verysensitive to the various elution buffers and/or thermal cycling that maybe required to cleave and/or dissociate the selected subset of a mixedpopulation of biological entities. In addition, an affinity-capturemethod utilizing a single enrichment column with a plurality ofreceptors is possible only provided that the bond linking one captureagent to a selected target subset is differentially dissociable from thebond linking the other capture agents to their respective, selectedtarget subsets, such that dissociation of the one capture agent from itstarget subset will not result in dissociation of another capture agentfrom its selected target subset. Thus, precise manipulation of thephysiochemical conditions (e.g., ionic strength, pH and concentration ofa particular cleaving reagent) is easier to achieve in individualmicrochannels of the parallel format. As a further advantage of thedevice and method of the invention for separating viable cells, is thatin the microfluidic platform large air bubbles—detrimental to recoveryof viable cells—do not form in the fluid pathway in which the cells aremanipulated.

A further significant advantage of the microfluidic devices and methodsof the present invention includes the integrated systems capabilitieswhich enable multiplexed cellular analyses to be performed on-line withthe cell purification process. For example, a portable self-containedmicrofluidics cartridge similar to that illustrated schematically inFIG. 27 may be employed in parallel with a conventional high gradientmagnetic separation (HGMS) device, as discussed below, for the rapid,quantitative and simultaneous measurement of a panel of tests to aid inthe diagnosis and treatment of human disease. As an alternative to theHGMS approach, a microfluidics based method and apparatus comprising amassively parallel channel configuration provides for economical, highthroughput cell purification combined with integrated cellulardiagnostics. In addition, this automated process is not laborious andtime consuming as are conventional cell isolation methods.

The present invention also broadly encompasses methods of usingintegrated microfluidic devices to deplete selected cells from a sample.High gradient magnetic separation (HGMS) has been used for the removalof magnetically labeled cells from suspensions of bone marrow,peripheral and/or cord blood cells. See, U.S. Pat. Nos. 5,514,340 and5,691,208, which are incorporated herein by reference in theirentireties. HGMS methods typically involve placing a filter of finemagnetizable wires in a strong magnetic field. High gradient magneticfields are produced around the wires, allowing the capture of even veryweakly magnetic particles upon the magnetizable wires.

Unlike the HGMS device described in U.S. Pat. No. 5,514,340, the presentinvention contemplates a microfluidic-based cell purification or cellpurging apparatus and method for recovering hematopoieticstem/progenitor cells from bone marrow, peripheral and cord blood and/orhematopoietic tissue for transplantation. Existing HGMS methods commonlyemploy a three stage process to achieve cell selection. Magneticallyconjugated antibodies are used to specifically target the desired cellsin a mixed population of cells. The noncaptured cells that have beentreated in the purification process can then be used for numerouspurposes, including, e.g., bone marrow/stem cell transplantation. Theintegrated chip-based cell-sorting device and method includes: 1) theflow-through incubation of selected cells and antibodies specific tocell surface antigens; 2) the addition of surface-activated magneticbeads which bind with the antibodies followed by another flow-throughincubation step; 3) application of a magnetic field for the affinitycapture of the bead-antibody-cell complex; and 4) the magnetic releaseof the complex or the chemical elution/thermal dissociation of theantibody-cell surface antigen bond. The device may be employed not onlyto deplete but also to further analyze the unwanted “selected” cells(e.g., T cells, tumor cells or oncotopes) from a mixed population.Analyses may include, but are not limited to, cell counting, cellstaining, cell sorting, cell lysis, genetic testing, competitive bindingand/or “sandwich” assays employing fluorescent or other like means fordetection. These assays have applications in immunodiagnostics,characterizing receptor-ligand affinity interactions and DNAhybridization reactions.

The release of cells from affinity matrices as described in U.S. Pat.No. 5,081,030, and multi-parameter cell separation using releasablecolloidal magnetic particles as described in, e.g., WO 96/31776 areincorporated herein by reference in their entireties.

The invention provides means for the automated electroactive control ofthe fluid circuitry without requiring the use of mechanical valves, asdescribed in U.S. Pat. No. 5,691,208. Electrokinetic pumping methods anddevices are described in, e.g., U.S. Ser. No. 08/615,642, filed Mar. 13,1996 now U.S. Pat. No. 5,750,015 the disclosure of which is herebyincorporated herein by reference in its entirety.

Monoclonal antibodies that recognize a stage-specific antigen orimmature human marrow cells and/or pluripotent lymphohematopoietic stemcells may be employed as described in, e.g., U.S. Pat. No. 4,714,680,which is incorporated herein by reference in its entirety.

As will be appreciated, where three or more outlet reservoirs areprovided, as for example is shown in FIG. 20, above, affinity captureand release can be effected, where one of the downstream reservoirscollects the purified or processed sample mixture. To provide theintroduction of the selected second or competing binding pair member torelease the bound entities of interest, additional input reservoirsupstream from the enrichment channel or affinity zone can be provided,as shown for example in FIG. 14.

The device of the invention may be used to deplete selected cells from asample, such as cells which express cell surface antigens recognized byantibodies, preferably monoclonal antibodies. In one embodiment of theinvention the method is used to deplete selected cells from cellsuspensions obtained from blood and bone marrow. In particular, themethod may be used to deplete tumor cells from bone marrow or bloodsamples harvested for autologous transplantation, or deplete Tlymphocytes from bone marrow or blood samples harvested for allogeneictransplantation. The device of the invention may also be used to removevirus particles from a sample.

The device and methods of the invention may be used in the processing ofbiological samples including bone marrow, cord blood and whole blood.

The device and methods of the invention are preferably used to depleteor purge tumor cells or T lymphocytes from samples to preparehematopoietic cell preparations for use in transplantation as well asother therapeutic methods that are readily apparent to those of skill inthe art. For example, in the case of an autologous transplant, bonemarrow can be harvested from a patient suffering from lymphoma or othermalignancies, the sample may be substantially depleted of any tumorcells using the device and methods described herein, and the resultinghematopoietic cell preparation may be used in therapeutic methods. Bonemarrow or blood can also be harvested from a donor in the case of anallogenic transplant and depleted of T lymphocytes by the methodsdescribed herein.

Using the method of the invention it is possible to recover a highlypurified preparation of hematopoietic cells. In particular, ahematopoietic cell population containing greater than 50% of thehematopoietic cells present in the original sample, and which isdepleted of T lymphocytes or tumor cells in the original sample bygreater than 2 logarithms may be obtained. The hematopoietic cells inthe preparation are not coated with antibodies or modified making themhighly suitable for transplantation and other therapeutic uses that arereadily apparent to those of skill in the art.

The method and device of the invention may also be used to remove redblood cells from samples such as blood and bone marrow. Half of thevolume of normal blood consists of mature red blood cells. Typicallythese cells exceed nucleated cells by >100 fold. For many clinical andresearch applications, removal of red blood cells with higher recoveryof cells than conventional methods such as Ficoll-Hypaque densitycentrifugation.

In a particular application of the invention, samples may be processedusing the methods and device described herein for diagnostic flowcytometry of leukocyte subpopulations. For example, the methods may beused to prepare blood samples of patients infected with the Human ImmunoDeficiency (HIV) virus for monitoring Iymphocyte populations in suchpatients. Enumeration of the absolute numbers of leukocyte subpopulationby conventional immunofluorescence measurements and flow cytometry hasbeen complicated by the abundant presence of red blood cells inperipheral blood and consequently, such enumeration is most oftenderived from separate measurements of nucleated cells numbers andimmunophenotype. A variety of procedures have been proposed and are usedto remove red blood cells from blood for immunophenotypic measurementsbut these procedures are labor intensive and difficult to automate andin some cases the procedure itself may interfere with immunofluorescencemeasurements. In contrast, the present invention provides an efficientand direct method for removing red blood cells from blood samples thatcan readily be automated as no centrifugation or wash steps areinvolved.

Specific Examples of uses to which the invention may be put include:Depletion of CD3+ T cells from allogeneic bone marrow using the deviceof the invention for the prevention of graft versus host disease (GVHD);Isolation of hematopoietic progenitor cells and depletion of malignantcells in patients with B-lymphoid malignancies; Removal of CD45RA+lymphoma cells from bone marrow; Purging of breast cancer cells fromperipheral blood and bone marrow; Purification of CD34+ cells byimmunomagnetic removal of CD34− cells; Depletion of murine cells thatexpress lineage markers; Immunomagnetic removal of red blood cells;Cellular diagnostics—employing a microfluidic-based panel of tests;Isolation of fetal nucleated erythrocytes from maternal blood; Isolationof genetically modified hematopoietic stem cells and depletion ofmalignant cells of non-hematopoietic origin—as for gene therapy, forinstance; Isolation and enumeration of selected cell populations of thehematopoietic cell lineages; Graft engineering for transplant; Captureof DNA and subsequent selective release of DNA recognized by probes withspecific sequences; AFLP analysis; Solid-phase sample clean-up of DNAsequencing products employing immune release (desbiotin fluorophore);and others.

In some embodiments it may be desirable to combine one or more reagentswith the enriched fraction downstream from the intersection of thesecondary flowpath and the main electrophoretic flowpath. FIG. 20 is aflow diagram similar to one shown in FIG. 15. In FIG. 20 a reagentflowpath 300 carries a reagent (or reagents) from a reservoir 301 to themain electrophoretic flowpath 238, where the reagent can combine withand react with one or more analytes in the enriched fraction. And, aswill be appreciated, where the main electrophoretic flowpath is brancheddownstream from the intersection with the secondary electrophoreticflowpath, producing subfractions in the branches, each such downstreambranch can be provided with a reagent flowpath carrying reagent from areservoir. Such a configuration can provide either for replicatetreatment of the subfractions with a single reagent, or for treatment ofeach subfraction with a different reagent, or for simultaneous treatmentof subfractions with two or more reagents, each producing a particulardesired result upon interaction with the analyte(s) in the enrichedsubfraction.

FIGS. 21 and 22 are flow diagrams similar to those shown in FIGS. 16 and17, having multiple branched main electrophoretic flowpaths, each branchprovided with an affinity zone. In FIG. 21 reagent flowpath 300 carriesa reagent (or reagents) from a reservoir 301 to the main electrophoreticflowpath 238, where the reagent can combine with and react with one ormore analytes in the enriched fraction. In this embodiment, because thereagent flowpath 300 intersects the main electrophoretic flowpath 238 ata point upstream from the first bifurcation, the reagent supplied byreservoir 301 effects a replicate treatment of all the subfractions thatare treated on the downstream branches and detected in the respectiveaffinity zones. In FIG. 22, each of the downstream branches of the mainelectrophoretic flowpath 238 is provided with reagent flowpath (302,304, 306, 308) each carrying a reagent from a separate reagent reservoir(303, 305, 307, 309). Such a configuration can provide for differenttreatment of the subfractions, for example, providing independentstringency control of parallel hybridization zones.

For example, devices providing flowpaths as in any of FIGS. 18 through22, or a combination of these, can be used for DNA profiling. Morespecifically, for example, restriction fragment polymorphism (“RFLP”)analysis can be carried out by employing a plurality of differentsingle-locus RFLP probes in reservoirs 303, 305, 307 and 309 as shown inFIG. 22. By running a large number of probes in parallel, the resultingdistribution of alleles should yield a rapid and representative DNAprofile, while significantly minimizing the possibility of randommatches.

The subject devices may be used in a variety of applications, where oneor more electric fields are applied to a medium to move entities throughthe medium. Representative applications include electrophoreticseparation applications, nucleic acid hybridization, ligand binding,preparation applications, sequencing applications, synthesisapplications, analyte identification applications, including clinical,environmental, quality control applications, and the like. Thus,depending on the particular application a variety of different fluidsamples may be introduced into the subject device, where representativesamples include bodily fluids, environmental fluid samples, e.g., waterand the like, or other fluid samples in which the identification and/orisolation of a particular analyte is desired. Depending on theparticular application, a variety of different analytes may be ofinterest, including drugs, toxins, naturally occurring compounds such aspeptides and nucleic acids, proteins, glycoproteins, organic andinorganic ions, steroids, and the like. Of particular interest is theuse of the subject devices in clinical applications, where the samplesthat may be analyzed include blood, urine, plasma, cerebrospinal fluid,tears, nasal or ear discharge, tissue lysate, saliva, ocular scratches,fine needle biopsies, and the like, where the sample may or may not needto be retreated, i.e., combined with a solvent to decrease viscosity,decrease ionic strength, or increase solubility or buffer to a specificpH, and the like, prior to introduction into the device. For clinicalapplications, analytes of interest include anions, cations, smallorganic molecules including metabolites of drugs or xenobiotics,peptides, proteins, glycoproteins, oligosaccharides, oligonucleotides,DNA, RNA, lipids, steroids, cholesterols, and the like.

The following examples are offered by way of illustration and not by wayof limitation.

EXAMPLE 1

High Efficiency Separation of Organic Analytes in an Aqueous Sample.

A card as shown in FIG. 4 is used in the separation of organic analytesin an aqueous sample as follows in conjunction with a device thatprovides for the application of appropriate electric fields throughintroduction of electrodes into each reservoir of the card and providesfor a means of detecting analyte as it passes through detection region65. In Card 50, the enrichment channel 62 comprises porous beads coatedwith a C-18 phase, while the reservoirs and channels, except for thewaste reservoir, comprise 20 millimolar borate buffer. A 100 ml aqueoussample is injected into enrichment channel 62 through interface 66.Substantially all of the organic analyte in the sample reversibly bindsto the C18 coated porous beads, while the remaining sample componentsflow out of enrichment channel 62 into waste reservoir 63. 10 ml of anelution buffer (90% methanol/10% 20 millimolar borate buffer pH 8.3) arethen introduced into the enrichment channel 62 through interface 66,whereby the reversibly bound organic analyte becomes free in the elutionbuffer. Because of the small volume of elution buffer employed, theconcentration of analyte in the volume of elution buffer as compared tothe analyte concentration in the original sample is increased 100 to1000 times. The seals over reservoirs 57 and 56 are then removed and anelectric field is applied between electrodes 61 and 60, causing bufferpresent in 57 to move towards 56, where movement of the buffer frontmoves the elution plug comprising the concentrated analyte tointersection 51. A voltage gradient is then applied between electrodes58 and 59, causing the narrow band of analyte present in the volume ofelution buffer to move through separation channel 52, yielding highefficiency separation of the organic analytes.

The above experiment is also performed in a modified version of thedevice depicted in FIG. 4. In the modified device, in addition toreservoir 57, the device comprises an elution buffer reservoir also influid communication with the enrichment channel 62. In this experiment,sample is introduced into enrichment channel 62, whereby the organicanalytes present in the elution buffer reversibly bind to the C18 phasecoated beads present in the enrichment channel. An electric field isapplied between an electrode present in the elution buffer reservoir andelectrode 60 for a limited period of time sufficient to cause 10 ml ofelution buffer to migrate through the enrichment channel and release anyreversibly bound organic analyte. After the elution buffer has movedinto the enrichment channel, a voltage gradient is then applied betweenelectrodes 61 and 60, resulting in the movement of buffer from 57 to 56,which carries the defined volume of organic analyte comprising elutionbuffer to intersection 51, as described above.

EXAMPLE 2

Sample enrichment employing paramagnetic beads for enrichment within anintegrated microfluidic device.

Experimental protocols based on biomagnetic separation methods areprovided as embodiments of the current invention. In a microfluidicdevice configured generally as illustrated in FIG. 23, and describedwith reference thereto, a crude sample composed of a particular targetis treated using magnetic beads, coated with an affinity medium, tocapture a target having a binding affinity for the specific affinitymedium. Such magnetic beads are marketed, for example, by Dynal, Inc.New York, under the name Dynabeads®. Dynabeads are superparamagnetic,monodispersed polystyrene microspheres coated with antibodies or otherbinding moieties that selectively bind to a target, which may be orinclude cells, genes, bacteria, or other biomolecules. Thetarget-Dynabead complex is then isolated using a magnet. The resultingbiomagnetic separation procedure is simple, rapid and reliable, wherebythe Dynabeads serve as a generic enrichment medium for isolatingspecific targets from complex heterogeneous biological mixtures. Suchmagnetic enrichment media may be employed according to the invention ina wide variety of applications involving cell biology, molecularbiology, HLA tissue typing, and microbiology, for example. Twoillustrative examples are provided here, specifically, methods for DNApurification and cell isolation.

First, the microchannel-based device is generally described, and thenthe method of employing Dynal beads for biomagnetic separation isgenerally described.

The integrated microfluidic device, as shown by way of example in FIG.23, includes a main electrophoretic flowpath 394 coupled to anenrichment channel 382, which includes a solid phase extraction (SPE)chamber 380, and which is connected to downstream waste reservoir 391.The main electrophoretic flowpath, which consists of an enriched-sampledetection region 393 and fluid outlet reservoir 395, joins the secondaryelectrophoretic flowpath 384 at a T intersection 388. In thisconfiguration, sample handling is achieved electrokinetically bycontrolling the electric potential across the appropriate electrodesplaced within the inlet reservoirs for the wash 373 and elution 375buffers and outlet reservoirs 391 and 395.

The Dynabeads and then the sample of interest are introduced into thedevice through injection inlet ports 379 and 377, respectively. Withinthe enrichment chamber, the heterogeneous suspension of sample andDynabeads specific for a given target incubate allowing the Dynabeads tobind the target by specific absorption to the particular capturemoieties on the surface of the beads. Immobilization of the target-beadcomplex to the side of enrichment chamber 380 is achieved magnetically.This is possible manually by placing a rare earth permanent magnetadjacent to the enrichment chamber. In another embodiment of theinvention, an automated protocol employing electromagnetic means is usedto control the applied magnetic field imposed on the SPE chamber.

Upon completion of the magnetic immobilization step, a wash mediumcontained within wash buffer reservoir 373 can be moved via pathway 374into and through enrichment channel 380. During sample rinsing, thewaste fluid passes out from the enrichment chamber 380 by way of theelectrophoretic flow path 384, then past the T intersection 388 and awaythrough the discharge outlet 392 to waste reservoir 391. Thus, thesupernatant from the wash steps is removed from the system withouthaving to pass the waste through the main electrophoretic channel 394.This embodiment of the invention affords an advantageous means forisolating and enriching the target biomolecule from a crude samplewithout first contaminating the detection region 393.

EXAMPLE 3

DNA purification from whole blood.

An experimental method employing an electrophoretic microdevice asschematically represented in FIG. 23 is provided in which Dynal®biomagnetic beads are used as an enrichment medium for extracting andpurifying genomic DNA from whole blood. The source of blood may be asmall dried forensic sample on a slide (e.g., on the order of ananogram), an aliquot of freshly drawn arterial blood (as small as 10ml) or bone marrow (approximately 5 ml). A protocol amenable to rapidDNA isolation and elution will be provided for the purpose ofdemonstrating an automated procedure for treating whole blood on-boardthe device of FIG. 23 so as to yield aliquots of DNA for amplificationand analysis or for direct analysis without amplification. The processincludes the following steps:

1. reagent and sample loading;

2. cell lysis/DNA capture;

3. repetitive DNA washes; and

4) DNA elution.

Each of these steps will now be discussed in more detail.

For this embodiment in which commercially packaged reagents are beingused, loading of the biomagnetic separation media, lysis solution andsample is achieved by means of specially designed injection ports toaccommodate differences in the reagents and sample. The Dynal DIRECTÔreagents, which include the lysis solution and magnetic beads, are firstinjected directly into the solid phase extraction chamber 380 via manualinjection port 379, followed by manual injection of the blood sampleinto the SPE chamber via the injection port 377. Alternatively, othercommercial reagents may be used where the nuclei lysis solution andbeads are not packaged together as a kit but are instead suppliedseparately. In this case, a lysis solution can be electrokineticallyloaded into the SPE chamber 380 from the inlet reservoir 371. Magneticbeads, supplied for example by Japan Synthetic Rubber, are next loadedeither from the injection port 379 or electrokinetically from inletreservoir 369. For the latter approach, the beads are confined to thechamber by electromagnetic capture, mechanical means (e.g., membrane,mesh screen, or agarose gel plug) placed just downstream of the SPEchamber in flowpath 384, or both. Once the chamber is filled with beadsand lysis solution, the DNA sample is added via the injection port 377,or electrokinetically via a sample inlet reservoir provided with anelectrode pair with an electrode downstream from the chamber.

Within the enrichment chamber, the blood sample, lysis solution andDynabeads are allowed to incubate for five minutes during which thecells are lysed. Released nucleic acids can then absorb to the capturemoieties immobilized on the surface of the microparticles to form aDNA-bead complex. To enhance cell lysis, mixing can be achieved by, forexample, arranging the supply channels so that the streams of beads,sample, and lysis solution merge. Mixing can be enhancedelectrokinetically by judicious control of the applied electric field.By periodically reversing the polarity of the electrodes placed in theinlet and outlet reservoirs 371 and 391, respectively, it is possible toelectrokinetically move the blood-lysis buffer mixture in an oscillatorymanner within the SPE chamber. To increase further the mechanical shearapplied to the cells, aperture-like structures can be molded into theSPE chamber housing.

Following the magnetic isolation and capture of the DNA-bead complex atthe side of the SPE chamber, rinsing is achieved by electrokinetictransport of the wash buffer solution contained in reservoir 373 throughthe chamber and out to the waste reservoir 391. After this 45 secondrinse, the beads are resuspended into solution by releasing the magneticfield and then allowed to incubate for one minute in the wash buffer.Following the same protocol, rinsing is repeated two more times,allowing the cell lysate and supernatant from each of the wash steps tobe removed from the system without having to pass the waste, includingPCR inhibitors, through the main electrophoretic channel 394.

The final step of the purification process is DNA elution. Again, thecapture beads with bound DNA are immobilized electromagnetically beforethe elution buffer is electrokinetically transported from reservoir 373into the SPE chamber. To obtain quantitative elution, precisemanipulation of electrode potentials is necessary, not to allow thebuffer to pass through the chamber and thus prematurely wash away thepurified DNA. Alternatively, a plug of elution buffer may be moved intothe chamber by employing an injection cross (not shown in FIG. 23) asdescribed in D. Benvegnu et al. U.S. patent application Ser. No.08/878,447, filed Jun. 18, 1997 now U.S. Pat. No. 5,900,130. With theelution buffer in the SPE chamber, the beads are resuspended byreleasing the magnetic field and then allowed to incubate in the elutionbuffer for two minutes allowing for finite DNA desorption kinetics. Uponcompletion of DNA elution, the beads are immobilized electromagneticallyin the SP chamber and the purified DNA is electrokinetically injected asa plug into the main electrophoretic channel 394 for analysis. Thedetection region 395 can represent an elaborate microfluidic system (notshown in FIG. 23) which may be comprised of a plurality of microchannelsfor restriction enzyme digestion, blot hybridizations, includingSouthern and slot/dot blots, electrophoretic fragment sizing, andquantitative PCR analysis, among others. These embodiments of theinvention will not, however, be discussed further in this example.

In summary, the above protocol allows for isolation of PCR-readyaliquots of purified DNA in less than ten minutes and without userintervention once the crude sample is introduced to the microfluidicdevice. Other advantages of the method include the minute amount ofreagents that are consumed in a given experiment, in addition to notrequiring more labor intensive precipitation or centrifugation steps.ADD others.

EXAMPLE 4

Cell enrichment employing immunomagnetic isolation.

An experimental protocol where Dynal® biomagnetic beads are used as anenrichment medium for isolating cell targets is provided. The procedureis similar to that described above for DNA purification. As in example3, the target is selectively captured by beads coated with specificbinding moieties immobilized on the surface of the paramagneticmicroparticles. Dynabeads are available prepared in various forms, asfollows:

1. precoated with affinity purified monoclonal antibodies to many humancell markers, including T cells, T cell subsets, B cells, monocytes,stem cells, myeloid cells, leukocytes and HLA Class II positive cells;

2. coated with secondary antibodies to mouse, rat, or rabbitimmunoglobulins for the isolation of rodent B cells, T cells and T cellsubsets;

3. in uncoated or tosylactivated form for custom coating with any givenbiomolecule; or

4. in streptavidin-coated form for use with biotinylated antibodies.

In a microfluidic device configured generally as illustrated in FIG. 23,a heterogeneous suspension of cells is treated employing electrokineticand magnetic manipulation methods to prepare purified aliquots of cellsfor further processing and analysis. Biomagnetic separation is possiblemanually or in an automated format employing electromagnetic control ofthe magnetic field imposed on the SPE chamber. The following four stepprotocol is provided as a representative embodiment of the invention.

1. loading of target cells and reagents, including biomagneticseparation media: load the solution of magnetic beads into SPE chamber380, either directly via injection port 379, or electrokinetically fromthe inlet reservoir 371 containing solution of Dynal beads specific to agiven target; or add sample directly to SPE chamber filled with solutionof Dynabeads by means of sample injection port 377.

2. cell capture employing Dynabeads capable of binding specific target:

allow sample and beads to incubate for 2.5 minutes within the SPEchamber, enhance adsorption by employing an electrokinetic mixing step,target cells bind to Dynabeads to form target-bead complex.

3. target cell wash by immobilizing the bead-target cell complex:

electromagnetically immobilize capture beads that contain the boundtarget, rinse with wash buffer solution by electrokinetic manipulation:

remove supernatant by controlling electrode potentials so as to passwash buffer from inlet reservoir 373 through the SP chamber to wasteoutlet 391,

stop the flow after 45 seconds and resuspend target-bead complex intosolution by releasing magnetic field,

incubate the target-bead complex in wash buffer for one minute,

repeat above wash steps two more times.

4. target cell elution employing Dynal's DETACHaBEADÔ reagents:

immobilize capture beads electromagnetically, load the DETACHaBEADÔsolution into SP chamber 380:

electrokinetically move the Dynal antibody-based reagent from theelution buffer reservoir 373 by manipulation of electrode potentials toavoid allowing the elution buffer to pass through the chamber, or,alternatively, an injection cross (not shown in FIG. 22) can be used toinject a plug of elution buffer into the SP chamber,

resuspend beads by releasing magnetic field,

incubate suspended beads in elution buffer for two minutes to allow forfinite desorption kinetics,

upon completion of target elution, immobilize beads electromagnetically

isolated target cells can be electrokinetically transported from the SPEchamber into the main electrophoretic channel for further treatment andanalysis.

Cell separations employing microfluidic devices and methods provide acost-effective alternative to conventional flow cytometry techniques. Inaddition, when combined with biomagnetic separation technology,microfluidic approaches enable cell enrichment and detection that yieldincreased sensitivity and reduced background noise. Microfluidic-basedmagnetic isolation methods subject the target substances to minimalstress, and can accordingly leave cells intact and viable, ready fordirect use in reverse transcription coupled with polymerase chainreaction amplification (RT-PCR). Microfluidic-based methods employ nophenol extractions, ethanol precipitations, or centrifugations, andemploy few toxic reagents. Separations are provided without the use ofexpensive equipment and are highly scalable.

EXAMPLE 5

Tools for Cost Effective Disease Management

As gene therapies move from the bench to the bedside, therapeutics anddiagnostics will become more intimately interlinked. Consequently,monitoring the efficacy of DNA-based pharmaceuticals usingbioinstruments at the bedside will become crucial to insuring thesuccess of these treatments. More specifically, a microfluidic-baseddevice for integrating cell collection and isolation processes withemerging molecular methods for DNA amplification and detection holdgreat promise for addressing this market need. Thus by combining methodsas described in this application (particularly examples 3 and 4), it ispossible to have in one analytical instrument the capability ofcost-efficient disease prognosis and monitoring for helping thephysician evaluate the appropriateness of a given genetic therapy. Sucheffective disease management strategies, in addition to otherpharmacogenetic approaches, have the potential for widespread use as thepost-genomic era rapidly approaches.

For the purpose of illustrating this embodiment of the invention, asystem for managing blood-based diseases will be presented.

For background purposes, inherited blood disorders are the most commongenetic diseases affecting humans. The World Health Organizationestimates that about 5% of the world's population are carriers ofdifferent types of hemoglobin disorders and that about 300,000 new casesare diagnosed each year. Sickle cell anemia and, b-thallasemia are thetwo most common hemoglobinopathies that may be treated using genetherapies.

Of particular interest in treating the hemoglobinopathies, as well asmonitoring the progress of their treatment, is the collection andisolation of hematopoietic stem cells. Employing the microfluidic deviceas shown in FIG. 22, when combined with the use of Dynal reagents forhuman hematopoietic progenitor cell selection as described in Example 4,a rapid and simple-to-use method for achieving the desired stem cellisolation is possible. For example, 1 ml of Dynabeads M-450 CD34 willisolate approximately 8×10⁷ cells. 100 ml (one unit) of DETACHaBEAD CD34is used to detach 4×10⁷ (100 ml) Dynabeads M-450 CD34. Cells isolatedwith this Progenitor Cell Selection System are pure (95% from bonemarrow, 90% from peripheral and cord blood) and phenotypicallyunaltered. On the same device, DNA analysis, including gene expressionmonitoring, is possible employing molecular genetic methods once thestem cells are isolated and then lysed. Thus, microfluidic-basedbioanalytical devices and methods, as described in this embodiment ofthe invention, should prove to be invaluable tools for diseasemanagement at this emerging molecular medicine and diagnosticsinterface.

EXAMPLE 6

Solid-phase isolation and enrichment

Solid phase extraction (SP) of a particular target from a heterogeneousmixture is achieved in the following embodiment of the invention byemploying the selective surface properties of target-specificmicroparticles and mechanical means for retention of the beads withinthe SP chamber. Although biomagnetic separation methods are currentlyattractive because commercial reagents are readily available for a widevariety of bioresearch applications, other non-magneticmicrofluidic-based approaches are possible for achieving comparableseparations. In similar embodiments to those provided above, solid phaseenrichment in a microfluidic format is presented. Beads withtarget-specific binding moieties can be retained within the enrichmentchamber utilizing mechanical means, including filtration membranes ormesh screens. In addition, an agarose gel may be injected (from thewaste reservoir 391 prior to the experiment) into channel 384 at theoutlet of the enrichment chamber 380 to prevent the beads from escaping,yet allowing the wash and elution buffers to pass through the highlyporous media. Thus, each of the embodiments described in Example 2 fortarget isolation and purification from complex mixtures may be achieved,at least conceptually, without requiring the use of magnetic fields.

EXAMPLE 7

In this example affinity-binding capture and release is employed tocollect and then release and separate biological entities of interest ina sample.

Here the biological entity is bound to one member of an affinity bindingpair, and is captured in an enrichment zone by affinity binding with theother member on a solid support. The enriched captures biological entityis then released, for example, by competitive displacement of thebinding pair by a binding pair member having a higher affinity.

In particular, for example, the biological entity of interest may beDNA. Generally, the method proceed as follows. One member of an affinitybinding pair is attached at the 5′ end of a selected oligonucleotidesequencing primer, which may be about 10-30 bases in length, usuallyabout 15-25 bases, or about 20 bases in length, to form a functionalizedprimer. The DNA of interest is combined with the functionalized primerin the presence of nucleotides under conditions favoring extension ofthe primer to form DNAs, complementary to the DNA of interest, andamplifying specific portions of the DNA. A dye terminator can beemployed in the reaction to provide a chromophore for fluorescencedetection of the amplified DNA portions. Each resulting amplified DNAhas a functional group at the 5′ end of each strand, and carries thechromophore. This sequencing reaction can be conducted outside thedevice, and the amplified DNA can be introduced to the enrichmentchannel by way of an inlet port; or the reaction can be conducted on thedevice itself.

The other member of the binding pair is then attached to a solidsurface, so that when the functionalized DNAs are brought into contactwith the solid surface under conditions favoring affinity binding of thebinding pair members, the DNAs are captured on the solid phase.According to the invention, the solid phase may be particles or beads,which can themselves be manipulated into, within, and out from thechannels or chambers of the device.

Release of the captured DNAs is then effected by introducing a bindingpair member that has a significantly higher affinity, with the resultthat it displaces either the binding pair member on the functionalizedDNAs, or the binding pair member on the solid support. This results infreeing the DNAs of interest, which can then flow out from theenrichment channel to a separation channel.

Any of a variety of affinity binding pairs may be used. For example, anavidin-biotin system may be employed. Avidin is attached to the solidsupport, and a modified biotin, having a significantly lower affinityfor avidin than unmodified biotin, is attached to the oligonucleotideprimer. Amplification is carried out, and then the amplified DNAs arecaptured in the device by binding of the modified biotin to the avidinon the solid support. Then release of the DNAs is effected byintroducing biotin into the enrichment channel to displace the modifiedbiotin, and the DNAs are moved out from the enrichment channel.

In an illustrative example, the functionalized oligonucleotide primeris-the M13/pUC forward 23-base sequencing primer, with dethiobiotinattached at the 5′ end, to form:

dethiobiotin-5′-CCCAG TCACG ACGTT GTAAA ACG-3′ (SEQ ID NO:1)

A general method of attaching dethiobiotin molecule to anoligonucleotide is shown in FIG. 26. Briefly,N-hydroxysuccinimidodedliobiotin (K. Ilofmann et al. (1982),Biochemistry, Vol. 21, page 978) (0.1 mMole) was reacted with 5′Amino-modifier C6 T (Glen Research; 0.1 Mole) as shown FIG. 26, to formdethiobiotin. To prepare the dethiobiotin-functionalized primer, thedethiobiotin was introduced by using dethiobiotin amidite ([2] in FIG.26) in the last step of the oligonucleotide synthesis on a DNAsynthesizer. After cleavage from the solid support and removal of thebase protecting groups the dethiobiotin conjugated primers were used inthe sequencing reactions.

Following amplification the amplified DNAs include a dethiobiotinfunctional group at the end of each strand of DNA. Referring now to FIG.24, the DNA sequencing products in the sample can be added to sampleinlet port 437. A filter or membrane material may be located at thebottom of the port to restrict access of particulate matter from sampleenrichment medium 432 that is confined within sample enrichment channel431. Preferably, the channels making up the device are located within aplane of the device, while the sample is introduced into the device fromoutside the plane of the device (for example, from above), and thetreated sample and/or wastes may leave the enrichment zone from anydimension. In the embodiments shown in FIGS. 24 and 25 the treatedsample leaves the enrichment zone through the waste fluid outlet 433below the plane of the device. All the reservoirs 435, 436, 434, 438,440 contain buffer, while reservoir 435 additionally contains biotin inan amount in the range 10 mMolar to 1000 mMolar. The flow through theenrichment zone can be controlled by application of a pressure gradientbetween the inlet 437 and the waste fluid outlet 433. Alternatively, thesample can be migrated through the enrichment zone by application of anelectric field between the sample inlet and a waste fluid reservoir.Beads or particles are coated with the protein carboxyavidin, which hasa strong affinity for dethiobiotin, and therefore will selectivelyenrich that component of the sample. The enrichment zone can be rinsedby application of an electrical potential between reservoirs 436 andeither 433 or 438. Following capture of the DNA sample, biotin locatedin reservoir 435 is moved through the enrichment zone by application ofan electric field between reservoirs 435 and 438. Biotin hassignificantly greater affinity for the carboxyevidin molecule than doesdethiobiotin (Kd=10⁻¹⁵ M for biotin, vs. 10⁻¹¹ M for dethiobiotin), andconsequently it displaces the DNA of interest from the beads in theenrichment zone. Injection of the released DNAs into the mainelectrophoresis channel 441 is performed by switching the electric fieldfor about 5 seconds to reservoirs 435 and 440. This causes a portion ofthe released DNA to migrate into the separation media within separationchannel 441. Changing the electric field between 434 and 440 results inseparation of the DNA in the main electrophoresis channel. Theseparation is detected at an optical detector 439.

In an alternative embodiment, differing in the arrangement of channelsdownstream from the enrichment channel, DNA is moved toward reservoir440 until a representative sampling is available at the inlet to themain separation channel 441. Injection of the DNA is accomplished bysimply switching the electric field to reservoirs 438 and 434 to performthe separation of DNA for detection at 439.

It is evident from the above results and discussion that convenient,integrated microchannel electrophoretic devices are disclosed whichprovide for significant advantages over currently available CE and MCEdevices. Because the subject devices comprise microchannels aselectrophoretic flowpaths, they provide for all of the benefits of CEand MCE devices, including rapid run times, the ability to use smallsample volumes, high separation efficiency, and the like. Since thesubject integrated devices comprise an enrichment channel, they can beemployed for the analysis of complex sample matrices comprising analyteconcentrations in the femtomolar to nanomolar range. However, because ofthe particular positional relationship of the enrichment channel and themain electrophoretic flowpath, the shortcomings of on-lineconfigurations, such as band broadening and the like, do not occur inthe subject devices. As the subject devices are integrated and compact,they are easy to handle and can be readily used with automated devices.Finally, with the appropriate selection of materials, the devices can befabricated so as to be disposable. Because of their versatility and thesensitivity they provide, the subject devices are suitable for use in awide variety of applications, including clinical electrophoretic assays.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1 1 23 DNA Oligonucleotide 1 cccagtcacg acgttgtaaa acg 23

What is claimed is:
 1. A method for nucleic acid sample clean-up usingan enrichment channel and a discharge outlet formed in a substrate, saidenrichment channel containing an enrichment medium and an affinitybinding pair having first and second binding members, said first bindingmember having an oligonucleotide moiety that is complementary in basesequence to a nucleic acid portion of said sample mixture, said secondbinding member being carried on a solid support in said medium, saidmethod comprising the steps of: introducing a nucleic acid mixturehaving a nucleic acid portion and a waste portion into the enrichmentchannel, combining the first binding member of the affinity binding pairwith the nucleic acid portion of at least some of the nucleic acidmixture to form bound entities between said nucleic acid portion andsaid moiety, contacting in the enrichment medium bound entities with thesecond binding member of the affinity binding pair, to capture at leasta part of the bound entities to form a captured nucleic acid portion,washing the captured nucleic acid portion to direct the waste portionand the nucleic acid portion, excluding the captured nucleic acidportion through said discharge outlet, and releasing the nucleic acidportion of said captured bound entities with a competitive displacingmember whose binding affinity for the second binding member is greaterthan that of the first binding member to yield a purified nucleic acidportion.
 2. The method according to claim 1 wherein the combining stepis performed after the introducing step.
 3. The method according toclaim 1 wherein the introducing step is performed after the combiningstep.
 4. The method according to claim 1 further comprising the step ofnucleic acid sequencing the purified nucleic acid portion.
 5. The methodaccording to claim 4 wherein the nucleic acid sequencing step includesthe step of dideoxy enzymatic chain-termination sequencing the purifiednucleic acid portion.
 6. The method according to claim 1 wherein thefirst binding member includes modified biotin having a lower affinitythan biotin to the second binding member.
 7. The method according toclaim 6 wherein the modified biotin is dethiobiotin.
 8. The methodaccording to claim 6 wherein the modified biotin is a dethiobiotinderivative.
 9. The method according to claim 1 wherein the secondbinding member includes an avidin-based protein.
 10. The methodaccording to claim 1 wherein the solid support includes a plurality ofmagnetic bodies.
 11. The method according to claim 1 wherein the solidsupport includes a plurality of paramagnetic bodies.
 12. The methodaccording to claim 1 wherein the competitive displacing member comprisesbiotin.
 13. The method according to claim 1 wherein the nucleic acidportion of the nucleic acid sample mixture includes a DNA template and asequencing primer.
 14. The method of claim 8, wherein the nucleic acidportion of the nucleic acid sample mixture includes a terminaldideoxynucleotide.
 15. The method according to claim 8 wherein thenucleic acid portion of the nucleic acid sample mixture includes adeoxynucleotide.
 16. The method according to claim 8 wherein said firstbinding member is dethiobiotin and wherein said second binding member isan avidin based protein and said competitive displacing member isbiotin, further comprising the step of performing PCR amplification onthe purified nucleic acid portion.